Contact with fungal and bacterial pathogens initiates a series of host responses, beginning with innate immunity. β-glucans are pathogen associated molecular patterns of fungi such as Candida albicans. Dectin-1 has been reported as the major receptor for β-glucans in studies done mostly on macrophages and dendritic cells, but not on keratinocytes. Here, we studied the effects of β-glucan on normal human epidermal keratinocytes (NHEK) from neonatal foreskin. We were also interested in investigating the effect of β-glucan on NHEK especially during conditions of skin damage and infection, which would allow deeper penetration of the fungi through impaired skin. We co-stimulated the cells with high calcium to induce keratinocyte differentiation, mimicking the uppermost layer of the epidermis; ATP, a danger signal released by UVB-exposed and damaged keratinocytes; and pathogen-associated compounds such as the TLR3 ligand, poly(I:C), found in viral infections; and the TLR4 ligand, LPS, found in bacterial infections. We also aimed to determine the possible role of dectin-1 in the process.

Subconfluent NHEK were stimulated with β-glucan 20 μg/ml or an equal amount of 0.3M NaOH, used to dissolve the β-glucan preparation. NHEK were pre-treated with either 0.05mM or 1.3 mM calcium for 24 hours, or ATP 250 uM, poly(I:C) 25 μg/ml, or LPS from E. coli or S. minnesota 10 ng/ml for 1 hour prior to β-glucan stimulation. Supernatants were collected at 12, 24 and 48 hours after stimulation with β-glucan. IL-8/CXC chemokine ligand 8, IL-1α, IL-6, IL-10, IL-12p40, IL-23, tumor necrosis factor-α, thymus and activation-regulated chemokine (TARC)/CC chemokine ligand 17, macrophage inflammatory protein (MIP)-3α/CC chemokine ligand 20 concentrations in the supernatants were measured by ELISA.

Cells were harvested and assessed for dectin-1 expression by reverse-transcription-PCR and flow cytometry analysis. Deectin-2 expression was also investigated using RT-PCR. Cell lysates of NHEK stimulated with β-glucan at 0, 5, 10, 15, 20, 30 and 60 minutes were used in immunoblotting to determine ERK, p38 MAPK, Akt or IKK α/β phosphorylation. Likewise, lysates of NHEK treated with high calcium, ATP or poly(I:C) alone were compared to lysates of NHEK pre-treated with these compounds and then stimulated with β-glucan. To assess ERK and p38 MAPK inhibition respectively, NHEK were pre-treated with the ERK inhibitor PD98059 or p38 MAPK inhibitor SB203580 prior to β-glucan stimulation. Supernatants were harvested after 24 hours to assess for IL-8 production, while cell lysates were prepared for immunoblotting.

NHEK responded to β-glucan, which induced significantly elevated levels of IL-8, and IL-6 compared to controls at 12, 24 and 48 hours. NaOH, reagent used to dissolve β-glucan, did not affect cytokine or chemokine secretion. No significant elevations were found in the other cytokines or chemokines tested.

β-glucan also induced higher levels of IL-8 secretion from well-differentiated NHEK compared to undifferentiated ones. No significant elevations in IL-1α and IL-6 were seen in well-differentiated NHEK treated with β-glucan. ATP upregulated IL-8 and IL-6 production in β-glucan-stimulated NHEK compared to β-glucan or ATP stimulation alone. IL-1α was not upregulated. The transient elevation of IL-1α noted in β-glucan-stimulated NHEK at 24 hours by ELISA was not found at the transcription level. However, poly(I:C) significantly upregulated IL-1α secretion in β-glucan-stimulated cells versus cells stimulated with β-glucan or poly(I:C) alone. No upregulation was noted for IL-8 or IL-6. No response to LPS was seen in NHEK, probably due to a very low expression of TLR4 in these cells.

We also found that NHEK expressed dectin-1 in both the mRNA and protein level by RT-PCR and flow cytometry analysis. Its cell surface expression was downregulated by β-glucan stimulation with flow cytometry analysis, signifying internalization of β-glucan by the cells. Dectin-2 was not expressed in NHEK.

The induction of ERK and p38 MAPK signaling pathways by fungal particles via the dectin-1 receptor had been studied using dendritic cells and macrophages. Thus we studied the induction of these two pathways in β-glucan-stimulated NHEK. Our results showed that β-glucan specifically induced the responses in NHEK via the ERK and p38 MAPK pathways. Although minimal activation of ERK and p38 MAPK was present when NHEK were treated with high calcium, ATP or poly(I:C) alone, addition of β-glucan significantly augmented ERK and p38 MAPK activation. Moreover, the addition of the ERK inhibitor PD98059 and the p38 MAPK inhibitor SB203580 effectively suppressed the IL-8 secretion in β-glucan-stimulated NHEK. Akt and IKKα/β phosphorylation was negative in NHEK stimulated with β-glucan.

In conclusion, high calcium, ATP and pathogen-derived components like poly(I:C) can augment inflammation in β-glucan-stimulated NHEK, leading to a rapid and effective host defense against cutaneous fungal infections. Dectin-1 expressed on NHEK may play an essential role in the responses to β-glucans by these cells, although further studies are needed to fully evaluate its role.

β-glucanはcandida albicansなどの真菌細胞壁を構成する高分子多糖である。真菌がヒト免疫系に認識されるにはこのβ-glucanが重要であるとされており、樹状細胞やマクロファージなどの貪食細胞に発現するC型レクチンのdectin-1はβ-glucanを認識して炎症を惹起することが知られている。一方、皮膚カンジダ症などで、真菌が経皮的に侵入する場合には、まず真菌を認識して、反応するのは表皮ケラチノサイトであり、実際に正常ヒトケラチノサイト(normal human epidermal keratinocytes:NHEK)をβ-glucan単刺激するとIL-8などのケモカイン産生が誘導されることがわかっている。そこで本研究は、ケラチノサイトの分化度、皮膚の掻痒、掻破に伴う細胞破壊、ウイルスの共存などの皮膚微小環境の影響を想定して、ケラチノサイトの分化を促進する高濃度カルシウム(1.3mM)、皮膚掻痒を引き起こすヒスタミン、細胞破壊に伴い細胞内から細胞外に放出されるATP、ウイルスの2本鎖RNAを模したpoly(I:C)存在下でNHEKをβ-glucan刺激して、サイトカインやケモカイン(IL-1Α、IL-6、IL-8)産生を検討し、下記の結果を得た。

1.NHEKは細胞表面にdectin-1を発現していることをフローサイトメトリーで確認した。

2.高濃度カルシウム、ヒスタミンはNHEKからのIL-8産生を促進し、ATPはIL-6、IL-8産生を促進し、poly(I:C)はIL-1Α産生を促進した。

3.β-glucan刺激はdectin-1のシグナル伝達下流分子のp44/42 MAPK (ERK1/2)とp38 MAPKを活性化することを免疫ブロットで確認した。

4.ERK阻害剤(PD98059)とp38キナーゼ阻害剤(SB203580)はいずれもβ-glucan刺激によるNHEKからのIL-8産生を有意に抑制した。

以上の結果から表皮ケラチノサイトに真菌が引き起こす炎症は細胞破壊やウイルスの共存などの微小環境の影響をうけることが示された。また、その反応はERK, p38 MAPK依存性であることから表皮ケラチノサイトによるβ-glucan認識にdectin-1が重要な役割を担っている可能性が示唆された。これらの結果は真菌による皮膚免疫の誘導システムに新たな知見を加えるものであり、学位授与に値すると考えられる。