潰瘍性大腸炎における粘膜細菌 Fusobacterium varium の病原性

F. varium is one of the elusive pathogenic factors in ulcerative colitis
大草敏史、佐藤信紘、岡安勲
順天堂大学医学部消化器内科、北里大学医学部病理【目的】我々は、潰瘍性大腸炎（UC）の病変粘膜から分離された Fusobacterium varium（F.

varium）が UC 患者の病変粘膜に特異的に付着していること、同菌が酪酸を産生し、その酪酸は Vero 細胞毒性を示し、その注腸により大腸潰瘍、大腸炎ができること。また、同菌に対する血清抗体価が UC 患者で有意に高値であることなどから、F.varium が UC の病原菌の一つではないかと報告してきた。今回、さらに病原性を明らかにするため、同菌の細胞侵入性や炎症性サイトカイン産生刺激作用を検討し、また、同菌を target とし、UC 患者に対して抗菌剤多剤併用療法を施行したので、その治療成績も含めて報告する。
【方法】1)大腸粘膜培養細胞 SW480 と F.varium(UC 粘膜分離株、ATCC8501), E.coli,H.pylori(ATCC43504), L.johnsonii(LC1), L.delbrueckii spp. bulgaricus(LB‑021001)を1.5X108/well と共培養（1,3 時間）して、細胞接着能、侵入能（回収培養菌量／負荷菌量％）とサイトカイン産生刺激能（IL‑8, IL‑6, IL‑18, MCP‑1)を検討した。
2）同菌に感受性の抗菌剤 3 剤（AMPC, TC, metronidazole)を 2 週間投与して、RCT を行いその治療効果を検討した。
【結果】1) F.varium は粘膜分離株と ATCC8501 共に、0.6‑2.9X104 CFU/mL と細胞侵入を認め、IL‑8 も 54‑146 pg/mL と産生されていた。また、IL‑18 は UC 粘膜分離株でのみ 37.2‑51.5pg/mL と産生されていた。IL‑6 と MCP‑1 についてはほとんど検出限界以下であった。接着能では、0.2‑4.2X104 と認められ、UC 粘膜分離株の 1 株で 34.5‑35.4 pg/mL と IL‑18 が検出された。一方、他の菌では、H.pylori で 1.0‑2.3X104 と細胞侵入性を認め IL‑8 も 54‑73.3と産生されたが、IL‑18 をはじめ、IL‑6 と MCP‑1 については検出限界以下であった。接着能も 1.5‑4.2 X104 と見られたが、サイトカイン産生は見られなかった。E.coli と L.bulgarisについては、接着能のみで、細胞侵入能は見られず、サイトカイン産生も認められなかった。LC‑1 菌については、細胞侵入能も接着能も 0.6‑3.9X104 と認められたが、サイトカイン産生はなかった。
2）対照群と比較すると、症状では抗菌剤投与群で 3 ヵ月後に有意な改善が認められ、12 ヶ月後も改善が持続していた。また、内視鏡所見、病理所見でも投与群で投与 3 ヵ月後、12ヶ月後と有意な改善を認めた。F.varium の血清抗体価と抗体染色による粘膜菌数についても投与群で 3 ヵ月後、12 ヵ月後と有意な低下が認められた。
【結論】F.varium が大腸粘膜に付着、侵入して炎症性サイトカインを産生させることが明らかとなった。また、同菌を target とした抗菌剤多剤併用療法が UC に対して有効であったことから、F.varium は UC の原因菌の１つであることが確認された。

F. varium is one of the elusive pathogenic factors in ulcerative colitis
Toshifumi Ohkusa, Isao Okayasu,* Nobuhiro Sato
Department of Gastroenterology, Juntendo University School of medicine, Tokyo and*Department of Pathology, Kitasato University School of Medicine, Kanagawa, Japan
Background & Aim: Recently, we proposed that intestinal bacteria in the adjacentintestinal mucosa (mucosal bacteria), and in particular, Fusobacterium varium might be
one of the elusive pathogenic factors in ulcerative colitis (UC). However, whether F.
varium cause inflammation of the intestinal mucosa has not been determined. In thisstudy, (1) we investigated bacterial penetration and the secretion of proinflammatory
cytokines in human colon epithelial cells in response to F.varium. Also, (2) weconducted a randomized controlled trial using antibiotics combination therapy in order
to confirm a causal relationship between F. varium and UC.
Methods: (1) Human colonic epithelial cells (SW480, ATCC) were cocultured withbacteria isolated from UC (F.varium, E.coli), H.pylori (ATCC 43504), and probiotics(Lactobacillus johnsonii (LC1), Nestle Japan Co., L.delbrueckii subsp. bulgaricus(LB-021001), Meiji Dairies Co., Japan). Cell cultures were infected with bacteria andresuspended in a 1:1 mixture of DMED and Ham’s F-12 medium containing 10% FBSat a bacterium/cell ratio of 100:1. The co-cultures further incubated for 1 h to allow
bacterial adhesion to cells, and the supernatants were collected for attachment assay.
After removal supernatants, the monolayers were washed four times with PBS, lysedwith water, and total cell-associated bacteria enumerated on agar plates. For invasion
assays, the incubation was continued for 3h to allow bacterial entry to occur. Afterremoval of the extracellular bacteria, the cultures were incubated for 1 h in the presenceof 50μg/mL of imipenem to kill the remaining extracellular bacteria. Subsequently, thesupernatants were removed and the cells were lysed with 0.1% SDS in isotonic saline,
and the number of internalized bacteria released was determined by culturing for 3 dayson agar plates. The IL-8, IL-6, IL-18 and MCP-1 concentrations in the supernatants
were determined with ELISA kits. (2) Twenty chronic, active UC patients with F.
varium infection were enrolled consecutively and were randomly assigned to receive
either amoxicillin, tetracycline and metronidazole per os for two weeks (treatmentgroup; n = 10), or no antibiotics (control group; n = 10). F. varium was sensitive to theantibiotics. Symptom assessment, endoscopic, and histological evaluations wereperformed blind before enrollment, at 3-5 months and 12-14 months after the treatment.
Serum immunoglobulins to F. varium were measured using ELISA.

Immunohistochemical detection of F. varium in biopsy specimens was performed withthe avidin-biotin complex method.
Results: (1) In invasion assays, F. varium was recovered from SW480 cells at 0.6-2.9×
104 CFU/mL. IL-8 concentrations after exposure to F.varium were 54-146 pg/mL andIL-18 concentrations after exposure to two F.varium strains from UC were 37.2-51.5
pg/mL. IL-6 and MCP-1 concentrations after exposure to all bacteria strains were below
0.150 and 31.3 pg/mL. In attachment assays, F. varium was recovered at 0.2-4.2×104
CFU/mL. Only IL-18 was detected as 34.5-35.4 pg/mL in one F.varium strains from UC.
In invasion assay, H. pylori were recovered from SW480 cells at 1.0-2.3×104 CFU/mL,and IL-8 concentrations after exposure to H.pylori were 54.0-73.3 pg/mL. In attachment
assays, H. pylori were recovered at 1.5-4.2×104 CFU/mL, however the cytokines werenot detected. E.coli and L. delbrueckii subsp. bulgaricus did not enter the SW480 cells,
and no cytokines were detected in the supernatants. L.johnsonii was recovered at 0.6-3.9
×104 CFU/mL, however the cytokines were not detected. (2) The clinical activity,endoscopic and histological scores in the treatment group decreased significantly at 3-5and 12-14 months after the end of treatment compared with those in the control group(p=0.001-0.036). The remission rate in the treatment group was higher than in control
group (p=0.037). In addition, the titers of antibody to F. varium and the F. varium
density in the mucosa decreased at both the short and long-term follow-ups in thetreatment group (p=0.0002-0.049).
Conclusion: F.varium isolated from UC invaded colonic epithelial cells and induced
IL-8 and IL-18 secretion. Antibiotics combination therapy against F. varium waseffective for ulcerative colitis. The number of mucosa-associated F. varium significantly
decreased after the treatment. Therefore, the results support the hypothesis that F.
varium is one of the causative agents of UC.