Supplementary Material for: High Molecular Weight Gingipains from <i>Porphyromonas gingivalis</i> Induce Cytokine Responses from Human Macrophage-Like Cells via a Nonproteolytic Mechanism

Periodontal disease is an oral inflammatory disease affecting the supporting structures of teeth. <i>Porphyromonas gingivalis</i>, a major pathogenic agent for the disease, expresses a number of virulence factors, including cysteine proteases called the gingipains. The arginine- and lysine-specific gingipains, HRgpA and Kgp, respectively, are expressed as high molecular weight forms containing both catalytic and adhesin subunits. We examined the expression pattern of cytokines and their receptors in differentiated macrophages following exposure to active and inactive forms of the gingipains, using a cDNA array, quantitative PCR and ELISA analysis. Amongst other pro-inflammatory cytokines, results from the cDNA array suggested that interleukin-1β, granulocyte-macrophage colony stimulatory factor and interferon-γ were upregulated after exposure of the macrophages to the gingipains. Quantitative PCR analysis substantiated these observations and indicated that active or inactive forms of the high molecular weight gingipains were able to upregulate expression of transcripts for these cytokines. The strongly enhanced production of interleukin-1β and granulocyte-macrophage colony stimulatory factor by differentiated macrophages in response to active or inactive forms of the high molecular weight gingipains was confirmed at the protein level by ELISA analysis. The results indicate that the adhesin subunits of the gingipains mediate strong upregulation of the expression of pro-inflammatory cytokines in macrophages.牙周病是一类侵袭牙齿支持组织的口腔炎症性疾病。牙龈卟啉单胞菌（Porphyromonas gingivalis）作为该疾病的主要致病菌，可表达多种毒力因子，其中包括被称为牙龈素的半胱氨酸蛋白酶。精氨酸特异性牙龈素与赖氨酸特异性牙龈素分别为HRgpA和Kgp，二者均以包含催化亚基与黏附素亚基的高分子量形式表达。本研究借助cDNA芯片、定量PCR及酶联免疫吸附试验（ELISA），探究了分化型巨噬细胞暴露于活性与非活性形式牙龈素后，其细胞因子及其受体的表达模式。在其他促炎细胞因子中，cDNA芯片检测结果显示，巨噬细胞暴露于牙龈素后，白细胞介素-1β、粒细胞-巨噬细胞集落刺激因子及γ干扰素的表达均出现上调。定量PCR分析验证了上述观测结果，并证实高分子量形式的活性与非活性牙龈素均可上调这些细胞因子的转录本表达。分化型巨噬细胞在响应高分子量形式的活性与非活性牙龈素时，其白细胞介素-1β与粒细胞-巨噬细胞集落刺激因子的产生显著增强，该结果在蛋白层面通过ELISA分析得到了验证。本研究结果表明，牙龈素的黏附素亚基可介导巨噬细胞中促炎细胞因子表达的显著上调。