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強皮症並びに関節リウマチの病態評価および病態解明に関する研究

蜷川, 慶太 北海道大学

2022.03.24

概要

全身性強皮症(SSc)は各臓器の慢性的な線維化を特徴とし、臓器病変の一つとして肺動脈性肺高血圧症(SSc-PAH)を呈し、予後は特に不良かつ難治である。診断の遅れや、左心疾患の合併で病態の評価が難しいことが原因に挙げられる。また、関節リウマチ(RA)は、慢性進行性の関節破壊を特徴とする自己免疫疾患である。滑膜線維芽細胞(SFs)は、高いオートファジー活性を有し、病態の中心的な役割を果たすが、オートファジーがどのような調整を受けているかは不明である。今回、我々はこれらを解明するべく研究を行った。

第一章境界型強皮症性肺高血圧症の評価方法に関する研究
【背景と目的】近年、SSc-PAH患者に対する早期の治療介入で予後が改善する可能性が示され、診断基準となる平均肺動脈圧(mPAP)の引き下げが提唱されている。本研究では、mPAP>20mmHgとなる強皮症患者の非侵襲的なスクリーニング法の作成を目的とした。

【対象と方法】2010~2018年に当院で右心カテーテル検査(RHC)を施行したSSc患者58名で、自己抗体、血漿BNP値、血清尿酸値、右軸偏位、努力性肺活量(FVC)一酸化炭素拡散能/(DLco)、三尖弁逆流速度を評価した。各マーカーでROC曲線を用いて、mPAP>20mmHgまたはmPAP≧25mmHgを予測する場合の正診能を比較した。また心臓核磁気共鳴画像法(MRI)を追加した場合の正診能を検討した。

【結果】mPAP≧25mmHgのときに匹敵して、FVC/DLcoと右軸偏位のmPAP>20mmHgを予測する特異度はそれぞれ72%、96%と高かった。それらのオッズ比に基づき重み付けをしてmPAP>20予測のアルゴリズムを作成すると、AUCが0.87、感度90%、特異度70%となった。心臓MRIを加えると、AUCが0.89、感度93%、特異度67%となった。

【結論】強皮症患者のmPAP>20mmHgを予測する新たなアルゴリズムを作成した。FVC/DLcoや右軸偏位の重み付け、心臓MRIの追加で予測精度が向上する可能性が示唆された。

第二章強皮症性肺動脈性肺高血圧症患者における左心疾患の評価
【背景と目的】SSc-PAHはしばしば左心疾患を合併し、治療抵抗性である。SScの心筋障害では拡張機能障害により、駆出率の保たれた心不全(HFpEF)として認められる。左心疾患による肺高血圧症(Group2PH)は肺動脈楔入圧(PAWP)≧15mmHgで定義されるが、早期発見のための新たなアルゴリズムが最近提唱された。本研究では、このアルゴリズムを用いてSSc-PAH患者における左心疾患の合併を評価した。

【対象と方法】2010~2019年に当院でRHCを受けた76人のSSc患者で、年齢、代謝・心血管疾患の有無、心電図、心臓超音波検査(TTE)を評価しGroup2PHらしさで、3群に分類した。臨床で実際に左心疾患を合併した患者数と比較し、アルゴリズムのSSc-PAHにおける妥当性を評価した。また、RHCやTTE、心臓MRIなどの画像モダリティーにおける各パラメーターをHFpEFを合併した患者としていない患者の間で、ROC曲線や多変量解析を用いて比較を行った。

【結果】76名中42名の患者はPH(mPAP>20mmHg)で、左心室駆出率が正常(≧50%)であった。うち10名がフラミンガム基準でもHFpEFと診断された。ROC曲線でHFpEFの合併を予想するカットオフを決定し、ロジスティック回帰分析でも評価すると、TTEと心臓MRIでそれぞれ評価した左心房径(LAD; cut off; 42mm, OR[95%CI]=14.5[2.55-82.2], p=0.003.)と左心室拡張末期容積指数(LVRMI; cut off: 57.1ml/m2, OR[95%CI]=20.0[1.83-218.0])は、PAWP(cut off: 19, OR[95%CI]=13.4[1.01-177.0])よりもHFpEFの合併症を検出するための予測値が有意に高かった。

【結論】TTEと心臓MRIによる形態学的評価は、PAWPによる血行動態学的な評価よりも、SScおよびPH患者における左心疾患の存在をより反映すると考えられる。

第三章関節リウマチの滑膜線維芽細胞におけるJAK-STAT経路によるオートファジー制御機序の解明
【背景と目的】我々の過去の研究で、オートファジーが亢進したRAのSFsではシトルリン化ビメンチン(cVIM)が増加し、MHCクラスII(HLA-DR)と相互作用し、オートファジーはRASFsにおける免疫学的特徴に密接に関係していると考えられた。しかし、詳細な機能などは不明な部分も多い。一方、JAK-STAT経路は多様なサイトカインの伝達に関わるため、オートファジーへの関与も考えられた。本研究では、SFsにおけるオートファジーの制御におけるJAK1の役割を調べた。

【対象と方法】当院で人工膝関節置換術を受けたRA患者の滑膜からSFsを分離・培養し、IFN-γや、飢餓刺激、プロテアソーム阻害薬であるMG-132でオートファジーを誘導した。JAK1の選択的阻害薬であるUpadacitinibの投与の有無で、LC3Bや、Beclin-1、ATG5、ATG7などのオートファジー関連遺伝子や蛋白質を、ウェスタンブロッティング(WB)法やリアルタイムPCR法で評価した。cVIMやHLA-DRの発現を免疫細胞染色法で評価し、これらの共発現を共免疫沈降法と近接ライゲーションアッセイで評価した。また、RASFsにおけるIL-6の産生を、培養上清のELASA法で評価した。

【結果】WB法では、飢餓刺激を加えたSFsにUpadacitinibを添加すると、LC-3II/βアクチン比は有意に低下し(p=0.016, n=7、リアルタイムPCR法でも、BECN1(p=0.01, n=6)、ATG5(p=0.03, n=6)、ATG7(p=0.02, n=6)と各遺伝子で有意に低下した。他の刺激条件でも同様であった。免疫細胞染色(n=1)では、IFN-γと飢餓刺激で刺激を行ったSFsではcVIMとHLA-DRの発現は亢進し、Upadacitinibの添加で低下した。cVIMとHLA-DRの共局在は共免疫沈降法において、飢餓刺激とIFN-γによる刺激で亢進し、Upadacitinibの添加で低下した(p=0.03, n=6)。近接ライゲーションアッセイでも同様であった。また、IFN-γによる刺激で培養上清中のIL-6は増加し(p=0.017, n=3)、Upadacitinibの添加で抑制された(p=0.02, n=5)。

【結論】オートファジーはJAK-STAT経路により調節され、その阻害でcVIMの生成やcVIMとHLA-DRとの共局在、IL-6の産生も制御された

SSc-PAHの早期例の検出にはFVC/DLcoや右軸偏位が有用で、左心疾患の早期例の検出には複数のモダリティーによる形態学的評価が重要であった。RAに関する研究の結果では、オートファジーがJAK-STAT経路による調節を受けていることが明らかとなり、病態の解明に大きく寄与するものと考えられる。

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参考文献

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