Combinational benefit of antihistamines and remdesivir for reducing SARS-CoV-2 replication and alleviating inflammation-induced lung injury in mice
摘要: 新冠肺炎（COVID-19）是由新型冠状病毒（SARS-CoV-2）感染导致的炎症相关疾病。抗病毒药物和抗炎药物联用有望在临床治疗取得有益的效果。我们最近证明，肥大细胞（Mast cell，MC）是SARS-CoV-2引发炎症的重要效应细胞。发现SARS-CoV-2刺突蛋白（Spike）通过诱导MC脱颗粒诱发肺泡上皮细胞炎症从而造成组织通透性破坏，并发现临床上治疗过敏性疾病的抗组胺药物可作为MC稳定剂，阻止Spike诱导的MC脱颗粒,进而抑制炎症发生和保护肺损伤。在该研究中，我们进一步阐述MC在SARS-CoV-2引发肺损伤中的作用，并评价抗组胺药物和抗病毒药物联用对病毒复制和炎症损伤的双重抑制效果。证明Spike诱导的MC脱颗粒可导致肺泡毛细血管损伤，而抗组胺药物预处理肺微血管内皮细胞，可防止细胞间紧密连接的破坏；特别重要一点，发现抗组胺药物HR1(histamine receptor)拮抗剂氯雷他定（Loratadine）和抗病毒药物瑞德西韦（Remdesivir）联用，可同时抑制SARS-CoV-2复制和缓解炎症造成的小鼠肺部损伤。该研究再次证明了MC在SARS-CoV-2诱导肺部炎症和导致肺损伤中的重要作用，并提出一种COVID-19抗病毒药物和抗炎药物联用治疗策略。Abstract: COVID-19 is an immune-mediated inflammatory disease caused by SARS-CoV-2 infection, the combination of anti-inflammatory and antiviral therapy is predicted to provide clinical benefits. We recently demonstrated that mast cells (MCs) are an essential mediator of SARS-CoV-2-initiated hyperinflammation. We also showed that spike protein-induced MC degranulation initiates alveolar epithelial inflammation for barrier disruption and suggested an off-label use of antihistamines as MC stabilizers to block degranulation and consequently suppress inflammation and prevent lung injury. In this study, we emphasized the essential role of MCs in SARS-CoV-2-induced lung lesions in vivo, and demonstrated the benefits of co-administration of antihistamines and antiviral drug remdesivir in SARS-CoV-2-infected mice. Specifically, SARS-CoV-2 spike protein-induced MC degranulation resulted in alveolar-capillary injury, while pretreatment of pulmonary microvascular endothelial cells with antihistamines prevented adhesion junction disruption; predictably, the combination of antiviral drug remdesivir with the antihistamine loratadine, a histamine receptor 1 (HR1) antagonist, dampened viral replication and inflammation, thereby greatly reducing lung injury. Our findings emphasize the crucial role of MCs in SARS-CoV-2-induced inflammation and lung injury and provide a feasible combination antiviral and anti-inflammatory therapy for COVID-19 treatment.
Figure 1. MC degranulation disrupts VE-cadherin in HULEC-5a cells
A–C: VE-cadherin expression observed by fluorescence microscopy. HULEC-5a cells (1×105) were seeded on a coverslip to form a monolayer, then treated with RBD-stimulated LAD2 cell culture supernatant (Deg. Supern.) (300 μL), or Medium (A) with RBD protein (2 μg/mL), or co-cultured with LAD2 cells (1×105) with or without RBD protein (B) for 48 h. In some groupss, Lor. (2.5 μg/mL), Eba. (1.5 μg/mL), or Ket. (4 μg/mL) was used to pretreat HULEC-5a cells for 2 h before stimulation (C). VE-cadherin was stained with specific antibodies and observed via fluorescence microscopy. Nucleus was stained with DAPI. Scale bar: 20 μm. D, E: VE-cadherin expression detected by flow cytometry. Cultured HULEC-5a cells were treated and immunostained as above, and VE-cadherin expression was detected by flow cytometry. Mean fluorescence intensity (MFI) was calculated. F, G: Spike/RBD-triggered MC activation for degranulation. LAD2 cells (3×105) were incubated with spike/RBD (2 μg/mL) at 37 °C for 1 h, fixed with 4% paraformaldehyde, permeabilized, immunostained with anti-avidin-FITC at 4 °C for 1 h, then analyzed by flow cytometry (F). Released histamines in cell culture supernatants were quantified by ELISA (G). Representative data are from four independent repeats.
Figure 2. MC degranulation induces inflammation in human microvascular endothelial cells
HULEC-5a cells were stimulated with RBD-treated LAD2 cell culture supernatants for 24 h. Total RNA was extracted, and transcriptome analysis was performed. A: Volcano plot of DEGs comparing LAD2/RBD supernatant-treated versus medium-treated groups. Symbols of up-regulated and down-regulated genes are shown. B: GO functional enrichment analysis of DEGs. Color bar indicates minus logarithm of q values, and bubble size indicates absolute gene counts enriched in GO terms. C: GSEA of distribution of gene sets related to inflammatory response and enrichment scores based on DEGs. D: Heatmaps showing relative expression level (left panel), fold-change (middle panel), and adjusted P-values (right panel) for cytokine/chemokine-related genes. E: Pretreatment with antihistamines blocks expression of pro-inflammatory factors. HULEC-5a cells were pretreated with Lor. (2.5 μg/mL), Eba. (1.5 μg/mL), or Ket. (4 μg/mL) for 2 h before stimulation. Cellular mRNA levels of S100A9, CXCL11, CCL7, CXCL10, IL-6, and ICAM-1 were quantified by qRT-PCR and normalized to gapdh mRNA. Representative data are from four independent repeats. F, G: IL-6 expression was detected by intracellular staining and flow cytometry. Three independent repeats are shown, MFI: Mean fluorescence intensity (G). Data are mean±standard deviation (SD). ***: P<0.001 indicates significant differences.
Figure 3. Combined remdesivir and Lor. treatment dampens SARS-CoV-2 replication and inflammation
A: Illustration of mouse treatments. BALB/c mice were infected intranasally with SARS-CoV-2 501Y.V2 strain (7.1×104 CCID50). In some mice, Lor. (10 mg/kg) was administered (i.p.) 1 day before infection, then given each day over the course of infection; remdesivir (25 mg/kg) was administered (i.p.) at the time of infection and used once daily until euthanasia. Each treatment group contained five mice; three mice without infection or drug treatment were used as mock controls. Lungs were collected at 3 dpi. B: Body weight was monitored. Mice were euthanized and lung lobes were harvested for analysis. C: Viral replication was monitored by quantifying expression of nucleocapsid gene. D: mRNA levels of IL-6, TNF-α, CCL5, CXCL11, CCL20, and ICMA-1 were quantified using qRT-PCR and normalized to gapdh mRNA. E: Frozen lung sections were immunostained with VE-cadherin to indicate pulmonary microvascular integrity. Nucleus was stained with DAPI. Scale bar: 50 μm. Data are mean±SD. *: P<0.05; **: P<0.01; ***: P<0.001.
Figure 4. Combined remdesivir and Lor. treatment reduces lung lesions in SARS-CoV-2-infected mice
Lungs from above treated BALB/c mice were collected at 3 dpi. A: Pathological lung sections with H&E staining. Scale bar: 100 μm. B: H&E score in lung sections. Data are mean±SD. *: P<0.05; ***: P<0.001.
Figure 5. Administration of Lor. or Lor./remdesivir blocks MC accumulation
A: Mock infection. B: BALB/c mice were infected intranasally with SARS-CoV-2 501Y.V2 strain (7.1×104 CCID50). C: Remdesivir (25 mg/kg) was administered (i.p.) at time of infection and used once daily until euthanasia. D, E: In some mice, Lor. (10 mg/kg) was administered (i.p.) 1 day before infection, then continued each day over the course of infection. Lungs were collected at 3 dpi. Toluidine blue staining of lung sections was used to observe MCs and their degranulation. Scale bar: 100 μm.
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