詹明修Ming-Shiou Jan魏郁玲Yu-Ling醫學研究所2026-10-289999-12-312007https://handle.ncl.edu.tw/11296/29db5zhttps://scholars.csmu.edu.tw/handle/123456789/35503雖然HLA-B27在僵直性脊椎炎 (Ankylosing Spondylitis, AS) 致病原中扮演著一個相當重要的角色，但HLA-B27對於AS其致病調節機制目前還未清楚，另外許多文獻指出克雷伯氏肺炎桿菌 (Klebsiella pneumoniae, KP) 與僵直性脊椎炎具有一定的相關性，但是對於KP造成AS的致病機轉至今還不明確。我們首先測試是否在KP處理之下會造成有表現HLA-B27的CA46 B淋巴母細胞株產生內質網壓力或是能夠調控HLA-B27的表現。實驗結果顯示並非莢膜或是熱殺死之KP，而是去莢膜化後的KP對於細胞株隨給予劑量及時間可以引起BiP/Grp78和Calreticulin的高度表現以及活化Caspase12，並促使HLA-B27 homodimers的形成且同時伴隨有B27重鏈蛋白質降解的情形產生。而這樣被去莢膜KP引起內質網壓力的表現同樣可發生在Jurkat T淋巴母細胞和U937單核球細胞。進一步研究証實在CA46 B淋巴母細胞中經由去莢膜KP刺激後，我們發現有p38活化以及ERK活性的現象，以salubrinal處理，證實可以提供部分的保護作用以對抗因細菌處理後造成內質網壓力所引起的細胞凋亡。另外，為了觀察在去莢膜KP處理下HLA-B27的表現是否會增加，我們將HLA-B27 promoter的報導載體轉染送進去U937單核球細胞株，結果顯示去莢膜化的KP會刺激HLA-B27 promoter驅動之基因表達。另外由臨床細胞觀察，AS病人的週邊血液單核球細胞 (Peripheral blood mononuclear cells, PBMC) 其細胞內BiP/Grp78的表現量明顯的比正常人還高，且病人的BiP表現量與臨床上使用來診斷疾病的BASFI數值是具有相關性的 (p=0.016)。此外，將去莢膜KP處理AS病人與正常人的PBMC一段時間後，AS病人細胞分泌IFN-gamma和TNF-alpha以及IL-10細胞激素的能力相對於正常人有明顯低下的情形。綜合這些結果顯示，提出可能的AS致病誘導假說：非莢膜之KP成分可刺激免疫細胞產生內質網壓力並改變HLA-B27表現構型，可能引發新細菌或自體抗原的呈獻，而啟動自體免疫反應，至於內質網壓力和HLA-B27的表現是否會利用共同的途徑去調控尚待釐清。Although HLA-B27 plays an important role in the etiology of ankylosing spondylitis (AS), the modulation of HLA-B27 expression in AS remains unclear. In addition, many studies have reported that Klebsiella pneumoniae is linked to AS, however the mechanism of pathogenesis remains unclear. First we examined whether the ER stress and HLA-B27 expression would be induced upon exposure of KP in HLA-B27- expressing B lymphoblast CA46 cells. We demonstrated that decapsulated KP, but not capsule polysaccharide fraction or heat-killed KP, triggered up-regulation of BiP/Grp78 and Calreticulin, Caspase12 activation, and enhancements of HLA-B27 homodimers formation and protein degradation in a dose- and time-dependent manner. Levels of ER stress induced by decapsulated KP treatment were also elevated in Jurkat T lymphoblasts and U937 monocytes. Further studies revealed that activation of p38 and suppression of ERK were simultaneously detected in CA46 cells upon decapsulated KP treatments. We subsequently found that a new pharmaceutical drug, salubrinal, provided a partial protection against KP-induced apoptosis via ER stress. On the other hand, we transfected U937 cells with pGL4.14-HLA-B27 promoter vector, and found that the reporter gene expression was enhanced upon decapsulated KP treatment. In addition, clinical investigation revealed that the intracellular BiP/Grp78 expressions in PBMC were higher in AS patients than that in normal individuals, and the BiP expressions were correlated to the clinical BASFI (Bath Ankylosing Spondylitis Function Index) values in diagnosis of AS disease (p=0.016). Moreover studies showed that the significant reductions of IFN-gamma, TNF-alpha, and IL-10 cytokines were detected in AS patients compared to normal individuals under decapsulated KP treatment. Taken together, we hypothesize that non-capsule components of KP might exhibit an autoimmunity induction by promoting bacterial or autoantigen presentation which results in the ER stress, HLA-B27 misfolding formation, protein degradation, and cell death. Whether KP induced ER stress and HLA-B27 expression via a common signal pathway require further investigation.thesis