Activation of the mTOR/HIF‐1α/VEGF axis promotes M1 macrophage polarization in non‐eosinophilic chronic rhinosinusitis with nasal polyps
Activation of the mTOR/HIF‐1α/VEGF axis promotes M1 macrophage polarization in non‐eosinophilic chronic rhinosinusitis with nasal polyps
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DOI：
10.1111/all.15050
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发表时间：
2021-08
期刊：
影响因子：
12.4
通讯作者：
B. Zhong;Jin-Tao Du;Feng Liu;Ya-Feng Liu;Shixi Liu;Lifeng Xie;Deyun 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;Lifeng Xie;Deyun Wang;L. Ba
慢性鼻窦炎伴鼻息肉（CRSwNP）是一种慢性鼻窦粘膜炎性疾病。嗜酸性粒细胞CRSwNP（eosCRSwNP）的特征在于与鼻息肉的相对低的嗜酸性粒细胞浸润（noneosCRSwNP）相比，主要的组织嗜酸性粒细胞浸润。目前，大多数研究都集中在eosCRSwNP的机制，然而，noneosCRSwNP在很大程度上是未探索的。低氧诱导因子1 α（hypoxiainducedfactor 1 α，HIF 1 α）是低氧条件下的一种稳定因子，在呼吸系统疾病中起重要作用。HIF 1 α在noneosCRSwNP的M1巨噬细胞中高度表达，并参与Th 1炎症反应。1哺乳动物雷帕霉素靶蛋白（mTOR）是一种丝氨酸/苏氨酸激酶，其信号通路调节细胞生理学的许多方面，如增殖、自噬和凋亡，先前已报道其参与HIF 1 α的调节。2然而，mTOR与HIF 1 α在CRSwNP中的关系及其病理机制尚不清楚。此外，HIF 1 α的重要下游因子血管内皮生长因子（VEGF）表达的调控机制也值得进一步研究。在此，我们试图确定mTOR、HIF 1 α和VEGF在noneosCRSwNP炎症中的作用。首先，免疫组织化学、蛋白质印迹和qPCR显示，noneosCRSwNP组织样品中的INOS、CD 86、pmTOR和HIF 1 α水平高于对照和eosCRSwNP样品中的水平（图1A）。CRSwNP组血管内皮生长因子（VEGF）水平高于对照组。此外，相关性分析表明，INOS和CD 86水平与mTOR、HIF 1 α和VEGF水平呈正相关，包括免疫组织化学和qPCR，尽管pmTOR的免疫组织化学水平与HIF 1 α和VEGF并不强相关（图S1）。接下来，我们发现pmTOR、HIF 1 α和VEGF与CD 86共染色，CD 86是noneosCRSwNP中M1巨噬细胞的特异性标志物（图2）。我们通过用iNOS（M1巨噬细胞的另一种标志物）共染色CD 86证实了M1巨噬细胞的特异性（图S2）。然后，我们将THP 1细胞分化为巨噬细胞，并用IFNγ和LPS刺激它们（图S3 A），这导致CD 86和iNOS的表达增加（图S3 B），以及TNFα和IL 12的mRNA水平增加（图S3 C）。这些结果表明在noneosCRSwNP样品中M1巨噬细胞极化。重要的是，随后的免疫荧光结果显示，在IFNγ和LPS刺激的巨噬细胞中，CD 86和pmTOR共染色的程度显著高于对照组。接下来，我们在刺激前用mTOR抑制剂雷帕霉素（RAPA）或HIF 1 α抑制剂KC 7 F2（KC）或VEGF抑制剂V1 Calbiochem（V1）处理巨噬细胞，以确认mTOR、HIF 1 α和VEGF与巨噬细胞极化之间的关系。IFNγ和LPS刺激后，巨噬细胞CD 86/pmTOR、CD 86/HIF 1 α和CD 86/VEGF共染表达均高于对照组。RAPA处理显著降低了共染色的⑶ 86/pmTOR的水平（图S4 A）。虽然KC处理也有降低作用，但不如RAPA处理明显。V1可抑制CD 86的表达，但对pmTOR和HIF 1 α无明显影响。此外，RAPA和KC均降低了共染色的CD 86/HIF 1 α和CD 86/VEGF的水平（图S4 B），而V1仅能抑制CD 86/VEGF的表达（图S5 A）。此外，我们还检测到INOS的表达，其与CD 86的表达相似（图S5 B）。此外，Western印迹和qPCR结果显示出相似的趋势（图S6）。既往研究表明mTOR可直接调节HIF 1 α的表达，进而调节中性粒细胞胞外陷阱的形成，3这可能解释了以中性粒细胞浸润为特征的noneosCRSwNP中mTOR和HIF 1 α的表达增加。事实上，mTOR通过HIF 1 α上调VEGF表达，这与我们的发现一致。一些报道表明，LPS和IFNγ在M1巨噬细胞极化过程中增加了HIF 1 α的表达。5同样，HIF 1 α水平的增加也促进了M1巨噬细胞的增殖。1有趣的是，mTOR 1的异常激活也可以直接促进M1的极化。6在我们的研究中，抑制mTOR、HIF 1 α和VEGF都降低了M1巨噬细胞的极化，抑制mTOR可下调HIF 1 α和VEGF的表达。而HIF 1 α和VEGF的抑制对mTOR的表达无明显影响。此外，抑制HIF 1 α可降低VEGF水平。因此，mTOR可能通过刺激noneosCRSwNP中HIF 1 α和VEGF的表达，促进M1巨噬细胞极化并导致炎症。
To the Editor, Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the sinonasal mucosa. Eosinophilic CRSwNP (eosCRSwNP) is characterized by predominant tissue eosinophilic infiltration as compared to relatively low eosinophilic infiltration (noneosCRSwNP) of the nasal polyps. At present, most studies have focused on the mechanisms underlying eosCRSwNP; however, noneosCRSwNP is largely unexplored. Hypoxiainduced factor1α (HIF1α) is a stable factor under hypoxic conditions that has been shown to play an important role in respiratory diseases. HIF1α has been shown to be highly expressed in M1 macrophages in noneosCRSwNP and to be involved in the Th1 inflammatory response.1 Mammalian target of rapamycin (mTOR) is a serine/threonine kinase with signaling pathway regulates many aspects of cell physiology, such as proliferation, autophagy, and apoptosis, which have been previously reported to be involved in the regulation of HIF1α.2 However, the relationship between mTOR and HIF1α in CRSwNP and its pathological mechanism remains unclear. In addition, the effect of regulatory mechanism on expression of vascular endothelial growth factor (VEGF), an important downstream factor of HIF1α, is also worthy of further investigation. Here, we sought to determine the role of mTOR, HIF1α, and VEGF in noneosCRSwNP inflammation. First, immunohistochemistry, Western blotting, and qPCR revealed that the levels of INOS, CD86, pmTOR, and HIF1α in noneosCRSwNP tissue samples were higher than those in control and eosCRSwNP samples (Figure 1A). Vascular endothelial growth factor (VEGF) in CRSwNP was higher than the control group. Moreover, a correlation analysis indicated that the levels of INOS and CD86 were positively correlated with those of mTOR, HIF1α, and VEGF, including immunohistochemistry and qPCR, although the immunohistochemical level of pmTOR was not strongly correlated with HIF1α and VEGF (Figure S1). Next, we found that pmTOR, HIF1α, and VEGF were costained with CD86, a marker specific for M1 macrophages in noneosCRSwNP (Figure 2). We confirmed the specificity of M1 macrophages by costaining CD86 with iNOS, another marker of M1 macrophages (Figure S2). Then, we differentiated THP1 cells into macrophages and stimulated them with IFNγ and LPS (Figure S3A), which resulted in increased expression of CD86 and iNOS (Figure S3B), and mRNA levels of TNFα and IL12 (Figure S3C). These results suggested M1 macrophage polarization in noneosCRSwNP samples. Importantly, subsequent immunofluorescence results showed that the degree of CD86 and pmTOR costaining in macrophages stimulated with IFNγ and LPS was significantly higher than that in the control group. We next treated macrophages either with the mTOR inhibitor rapamycin (RAPA) or the HIF1α inhibitor KC7F2 (KC) or the VEGF inhibitor V1Calbiochem (V1) prior to stimulation to confirm the relationship between mTOR, HIF1α, and VEGF and macrophage polarization. The levels of costained CD86/pmTOR, CD86/HIF1α, and CD86/VEGF were higher in the macrophages stimulated by IFNγ and LPS than in the control group. RAPA treatment significantly reduced the levels of costained CD86/pmTOR (Figure S4A). Although KC treatment also had a reducing effect, it was not as pronounced as that of RAPA treatment. In addition, V1 could inhibit the expression of CD86, but had no significant effect on pmTOR and HIF1α. Moreover, both RAPA and KC reduced the levels of costained CD86/HIF1α and CD86/VEGF (Figure S4B), while V1 can only inhibit the expression of CD86/VEGF (Figure S5A). In addition, we also detected the expression of INOS, which was similar to that of CD86 (Figure S5B). Furthermore, Western blot and qPCR results showed a similar trend (Figure S6). Previous studies have shown that mTOR can directly regulate the expression of HIF1α and further regulate neutrophil extracellular trap formation,3 which may explain the increased expression of mTOR and HIF1α in noneosCRSwNP characterized by neutrophil infiltration. Indeed, mTOR upregulates VEGF expression through HIF1α,4 which is consistent with our findings. Some reports have demonstrated that LPS and IFNγ increase HIF1α expression in the process of M1 macrophage polarization.5 Similarly, increased levels of HIF1α also promote the proliferation of M1 macrophages.1 Interestingly, the abnormal activation of mTORC1 can also directly promote M1 polarization.6 In our study, inhibition of mTOR, HIF1α, and VEGF all reduced the polarization of M1 macrophages, but inhibition of mTOR could downregulate the expression of HIF1α and VEGF. While inhibition of both HIF1α and VEGF showed no significant effect on the expression of mTOR. In addition, inhibition of HIF1α decreased VEGF levels. Therefore, mTOR may promote M1 macrophage polarization and lead to inflammation by stimulating HIF1α and VEGF expression in noneosCRSwNP.