鳥嘌呤交換因子H1在NLRP3發炎體調控中的角色

Abstract

先天免疫系統是身體抵抗病原體入侵的第一道防線。此系統產生各式的模式識別受體 (PRR) 針對不同的病源體相關物質模式 (PAMP) 來產生不同的免疫反應。先前研究發現NLR family pyrin domain containing 3 (NLRP3) 的活化不但會被微管網路所調控，而且需要乙醯化的α微管以提供最適合NLRP3和apoptosis-associated speck-like protein containing a CARD (ASC) 組合的位置。鳥嘌呤交換因子H1 (guanine exchange factor H1, GEF-H1) 是調節微管和肌動蛋白網路之間的交互作用最重要的分子，而且在COS-7細胞內過度表現GEF-H1會加強乙醯化α微管的產生。此外，GEF-H1已經被發現參與在位於細胞質中的PRR訊息傳遞中扮演重要的角色。由於NLRP3位於細胞質中而且乙醯化的α微管累積是的NLRP3活化的重要因子，所以我們想研究GEF-H1在NLRP3活化中所扮演的角色。 首先我們使用奈及利亞菌素 (nigericin) 及樊 (alum) 作為活化NLRP3的刺激物。結果顯示GEF-H1不影響 NLRP3發炎體活化所引起的介白素1β (IL-1β) 的分泌。由於介白素1β的分泌需要凋亡蛋白酶1 (caspase-1) 的自我切割而活化且同時會造成細胞凋亡，我們進一步發現GEF-H1並不影響凋亡蛋白酶1的活化與細胞凋亡的程度。此外，GEF-H1也不影響在NLRP3活化時，細胞內乙醯化α微管的累積。綜合上述結果，我們的研究證明GEF-H1在NLRP3活化是沒有角色的。 最後我們想瞭解GEF-H1是否能調節NLR family CARD domain containing 4 (NLRC4) 發炎體的活化。在缺少GEF-H1的細胞中，其IL-1β分泌和凋亡蛋白酶1的活化都是下降的。整體來說，GEF-H1不參與在NLRP3發炎體的活化中但可調節NLRC4發炎體活化。

Innate immune system is the first line of defense against invading pathogens or endogenous danger signals. In order to distinguish different pathogen-associated molecular patterns (PAMPs), immune system develops several pattern recognition receptors (PRRs) to generate specific immune responses. NLR family pyrin domain containing 3 (NLRP3), one of the cytoplasmic PRRs, is regulated by the microtubule network. The accumulation of acetylated α-tubulin is a key factor to create an optimal site for the assembly of NLRP3 and apoptosis-associated speck-like protein containing a CARD (ASC). Guanine exchange factor H1 (GEF-H1) is critical for the cross-talk between microtubule and actin networks. Furthermore, overexpression of GEF-H1 in COS-7 cells induces α-tubulin acetylation. It has been proven that GEF-H1 regulates cytoplasmic PRRs nucleotide binding oligomerization domain containing 1 (NOD1)-, nucleotide binding oligomerization domain containing 2 (NOD2)- and RIG-I like receptor (RLR)-mediated immune responses. Give NLRP3 is a cytoplasmic PRR and its activation requires tubulin acetylation, we investigate whether GEF-H1 is involved in NLRP3 activation. We first examined NLRP3-dependent IL-1β secretion and found that there was no significant difference between LPS-primed wild-type and GEF-H1-deficient bone marrow derived macrophages (BMDMs) incubated with nigericin. It is well known that IL-1β maturation requires caspase-1 autoprocessing. Therefore we examined caspase-1 maturation and found that GEF-H1 is dispensable in caspase-1 maturation in LPS-primed BMDM incubated with nigericin. Since caspase-1 autoprocessing leads to programmed cell death called pyroptosis, we then examined the cell viability. The result showed no viability difference between LPS-primed wild-type and GEF-H1-deficient BMDMs incubated with nigericin. Furthermore, we investigated α-tubulin acetylation after NLRP3 activation. The accumulation of acetylated α-tubulin was similar in LPS-primed GEF-H1-deficient BMDMs incubated with nigericin compared with wild-type BMDMs. These results implicated that GEF-H1 did not regulate the activation of NLRP3 inflammasome. Finally, we tested NLR family CARD domain containing 4 (NLRC4)-dependent IL-1β secretion and caspase-1 autoprocessing to determine whether GEF-H1 was involved in the activation of NLRC4. Compared to wild-type BMDMs, IL-1β secretion and caspase-1 autoprocessing were both reduced in LPS-primed GEF-H1-deficient BMDMs incubated with flagellin. This indicated that GEF-H1 may play a role in NLRC4 activation. Taken together, GEF-H1 is dispensable in the activation of NLRP3 inflammasome but is able to regulate NLRC4 inflammasome activation.