Hypoxia‐induced factor‐1α induces NLRP3 expression by M1 macrophages in noneosinophilic chronic rhinosinusitis with nasal polyps
Hypoxia‐induced factor‐1α induces NLRP3 expression by M1 macrophages in noneosinophilic chronic rhinosinusitis with nasal polyps
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DOI：
10.1111/all.14571
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发表时间：
2020-08
期刊：
影响因子：
12.4
通讯作者：
B. Zhong;Jin-Tao Du;Feng Liu;Ya-Feng Liu;Shixi Liu;Jie Zhang;P. Lin;Jiao Zhou;Jing Liu;H. H. Ong-H.;Tan Kai Sen;D. Wang;L. Ba
中科院分区：
文献类型：
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作者：
B. Zhong;Jin-Tao Du;Feng Liu;Ya-Feng Liu;Shixi Liu;Jie Zhang;P. Lin;Jiao Zhou;Jing Liu;H. H. Ong-H.;Tan Kai Sen;D. Wang;L. Ba
据编辑介绍，慢性鼻-鼻窦炎合并鼻息肉(CRSwNP)是一种广泛存在的鼻黏膜慢性炎症性疾病，可表现为不同的表型。嗜酸性CRSwNP(EosCRSwNP)通常在黏膜下表现为嗜酸性粒细胞聚集，而非eosCRSwNP与中性粒细胞浸润有关。1鉴于不同类型的CRSwNP，特别是非eosCRSwNP，可能有不同的炎症途径，因此有必要探讨非eosCRSwNP的机制，为CRSwNP的个体化治疗创造条件。近年来，缺氧诱导因子-1α(HIF-1α)被证明在上呼吸道炎症的启动和维持中起着关键作用。2先前的研究发现，与正常黏膜相比，HIF-1α促进慢性阻塞性肺疾病患者呼吸道上皮细胞MUC5AC的表达，促进黏液分泌和上皮细胞增殖。3含3个吡咯结构域的NOD样受体家族炎症小体是由NLRP3、AsC和Caspase-1组成的多蛋白复合体，也被发现参与了持久的呼吸道炎症。关于缺氧诱导因子-1α与炎症小体NLRP3在慢性肾功能衰竭发病机制中的相互作用研究较少。因此，本研究试图确定缺氧诱导因子-1α和NLRP3在促进CRSwNP炎症反应中的作用。通过对鼻息肉组织进行Western blotting，我们发现非eosCRSwNP组的HIF-1α、NLRP3、Caspase-1、Asc和磷酸化p38MAPK的蛋白水平显著高于对照组和eosCRSwNP组(图1A，B)。此外，信使核糖核酸水平也显示出类似的趋势(图1C)。值得注意的是，HIF-1α的表达水平与NLRP3炎症体(NLRP3、Caspase-1和Asc)及炎性细胞因子的表达水平相关(图1D)。此外，我们还发现Arg-1阳性的II型巨噬细胞主要见于eosCRSwNP，而iNOS阳性的I型巨噬细胞主要见于非eosCRSwNP。更重要的是，共定位的结果表明，在非eosCRSwNP中，HIF-1α与iNOS巨噬细胞共染色，而不是与Arg-1巨噬细胞共染色。此外，在非eosCRSwNP中，NLRP3也显示出与iNOS巨噬细胞类似的共染色。有趣的是，我们还发现，在非eosCRSwNP中，HIF-1α和iNOS的共同染色水平与NLRP3和iNOS共同染色的水平呈正相关(图2A)。为进一步证明iNOS巨噬细胞参与低氧诱导的非eosCRSwNP，用佛波酯(PMA)刺激THP-1细胞24小时转化为巨噬细胞，再用氯化钴(CoCl2)处理24小时模拟细胞缺氧。结果显示，iNOS在蛋白和mRNA水平均显著高于对照组。另一方面，Arg-1的水平没有明显变化，表明在低氧条件下有更多的iNOS阳性的I型巨噬细胞参与(图2B)。低氧条件下HIF-1MAPK、NLRP3、Caspase-1、Asc和磷酸化p38MAPK的蛋白和α水平也有升高趋势(图2C，D)。此外，免疫荧光显示低氧刺激后缺氧诱导因子-1α和NLRP3与iNOS的共染色增加(图2E)。为了评估缺氧诱导的HIF-1α在诱导巨噬细胞NLRP3炎症体激活中的作用，我们进行了类似的低氧刺激实验，同时通过siRNA介导的基因敲除来下调HIF-1α或NLRP3的表达。结果表明，在低氧条件下，随着HIF-1α和NLRP3蛋白水平的降低，Caspase-1和Asc蛋白水平也随之降低。同样，我们的结果也表明，磷酸化p38MAPK通路水平也降低(图S1a，C)。同时，我们还发现，siHIF-1α抑制NLEP3的水平，但反之亦然，这表明HIF-1α在NLRP3介导的炎性小体激活的上游发挥作用，在iNOS阳性的I型巨噬细胞中起激活作用。相应地，Caspase-1、Asc、IL-1β、IL-18、IL-6和IL-8mRNA水平均在抑制HIF-1α和NLRP3后下降(图S1B，D)。DOI：10.1111/all.14571
To the Editor, Chronic rhinosinusitis with nasal polyps (CRSwNP) is a widespread chronic inflammatory disease of the nasal mucosa and can manifest in different phenotypes. Eosinophilic CRSwNP (eosCRSwNP) usually exhibits eosinophil aggregation under the mucosa, while noneosCRSwNP is associated with neutrophil infiltration.1 Considering that different types of CRSwNP, especially non-eosCRSwNP, may have different inflammatory pathways, it is imperative to explore the mechanisms of non-eosCRSwNP for developing personalized treatments for CRSwNP management. In recent years, hypoxia-induced factor-1α (HIF-1α) has been shown to play a key role in initiating and maintaining inflammation in the upper respiratory tract.2 A previous study found that HIF-1α promoted the expression of MUC5AC in airway epithelial cells of chronic obstructive pulmonary diseases patients when compared with normal mucosa, promoting mucus secretion and epithelial cell proliferation.3 Pyrin domain containing 3 (NLRP3) inflammasome of NOD-like receptor family is a multi-protein complex composed of NLRP3, ASC, and caspase-1 that were also found to be involved in perpetuating airway inflammation.4 As of now, however, there are few studies on the interaction between HIF-1α and NLRP3 inflammasome in CRSwNP pathogenesis. Thus, the present study sought to determine the role of HIF-1α and NLRP3 in promoting inflammation in CRSwNP. By Western blotting on nasal polyps tissues, we found that HIF-1α, NLRP3, caspase-1, ASC, and phospho-p38 MAPK protein levels in non-eosCRSwNP were significantly higher than the controls and eosCRSwNP group (Figure 1A,B). In addition, mRNA levels also showed similar trends (Figure 1C). It is interesting to note that the mRNA level of HIF-1α was correlated with the mRNA level of NLRP3 inflammasome (NLRP3, caspase-1, and ASC) and inflammatory cytokines (Figure 1D). Additionally, we found that Arg-1-positive type II macrophage was mainly observed in eosCRSwNP, while the INOS-positive type I macrophage was observed mostly in non-eosCRSwNP. More importantly, the results of co-localization suggested that HIF-1α co-stained with INOS macrophages in non-eosCRSwNP rather than with Arg-1 macrophages. Furthermore, NLRP3 also showed similar co-staining with INOS macrophages, in non-eosCRSwNP. Interestingly, we also found that the levels of co-staining between HIF-1α and INOS were positively correlated with levels of NLRP3 and INOS co-staining in non-eosCRSwNP (Figure 2A). To further show INOS macrophage involvement in hypoxia-induced non-eosCRSwNP, THP-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 hours to transform into macrophages, which were then further treated with cobalt chloride (CoCl2) for 24 hours to simulate cellular hypoxia. The results showed that the INOS was significantly higher at protein and mRNA levels than that of the control group. On the other hand, the level of Arg-1 did not change significantly, indicating more INOS-positive type I macrophage involvement in hypoxic conditions (Figure 2B). Protein and mRNA levels also indicated that HIF-1α, NLRP3, caspase-1, ASC, and phospho-p38 MAPK showed elevated trends under hypoxic conditions (Figure 2C,D). In addition, immunofluorescence indicated that the co-staining of HIF-1α and NLRP3 with INOS increased after hypoxia stimulation by CoCl2 (Figure 2E). To assess the role of hypoxia-induced HIF-1α in inducing NLRP3 inflammasome activation in macrophages, a cell type frequently associated with nasal polyps, we performed similar hypoxia stimulation experiments while knocking down HIF-1α or NLRP3 expression with siRNA-mediated knockdown. The results showed that with a decrease in HIF-1α or NLRP3 protein level under hypoxic conditions, caspase-1 and ASC protein levels were reduced. Similarly, our results also suggested that phospho-p38 MAPK pathway level was also reduced (Figure S1A,C). Accompanying the reduction, we also found that siHIF-1α inhibited the levels of NLEP3, but not the other way around, indicating that HIF-1α functions upstream of the NLRP3-mediated inflammasome activation, serving to activate it in INOS-positive type I macrophages. Correspondingly, mRNA levels of caspase-1, ASC, IL-1β, IL-18, IL-6, and IL-8 were all decreased following suppression of HIF-1α and NLRP3 (Figure S1B,D). DOI: 10.1111/all.14571