BREAKING COVID-19 News! Study identifies RAGE As A Receptor For SARS-CoV-2 N Protein And It Also Mediates N Protein-induced Acute Lung Injury!

COVID-19 News: In the midst of the COVID-19 pandemic, breaking news from the Children's Hospital of Zhejiang University School of Medicine and the National Clinical Research Center for Child Health in Hangzhou, China, brings a ray of hope and a significant leap in our understanding of the novel coronavirus. A groundbreaking study has identified RAGE (Receptor for Advanced Glycation Endproducts) as a receptor for the SARS-CoV-2 N Protein, potentially offering new avenues for therapeutic interventions. This discovery not only sheds light on the virus's pathogenic mechanisms but also holds the potential to ameliorate the severe acute lung injuries associated with COVID-19.


RAGE mediated N-protein–induced acute lung injury. (A–C) WT C57BL/6 mice were administered intratracheally with His-tagged full-length N-protein (75 μg/mouse in 50 μl) or PBS. (A) BAL and serum samples were harvested at 24 hours. RAGE levels in the BAL and serum were determined by ELISA (n = 8). (B) Total RAGE mRNA expression in BAL cells was examined via qRT-PCR (n = 9). (C) RAGE expression in lung tissue was detected via immunohistochemistry (n = 3). Scale bars, 20 μm. (D–F) RAGE−/− mice with C57BL/6 background and WT C57BL/6 mice were treated with PBS or N-protein (75 μg/mouse in 50 μl) intratracheally. Lung tissue and BAL samples were collected at 24 hours after N-protein insult. (D) Tissue sections were stained with hematoxylin and eosin (n = 3). Scale bars, 50 μm. (E) Total protein, total cells, and neutrophils in the BAL were assayed to evaluate lung injury (n = 8). (F) Levels of IL-1β, IL-6, and TNF-α in the BAL were determined via ELISA (n = 8). Data are presented as mean ± SD. **P < 0.01 and ***P < 0.001. Student’s two-tailed t test (A and B). Two-way ANOVA with Tukey’s post hoc test (E and F).

The Background
The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had far-reaching consequences on public health and the global economy. Among its most severe complications are acute lung injury and acute respiratory distress syndrome (ARDS), which are associated with a high mortality rate as covered in past studies and COVID-19 News reports. The incidence of ARDS among COVID-19 non-survivors is approximately 90%, making it a significant cause of death in COVID-19 cases. ARDS, in both classic and COVID-19 forms, is characterized by increased endothelial permeability, infiltration of inflammatory cells, and elevated proinflammatory cytokines like IL-6, IL-8, and TNF-α.
 
A Deeper Look into SARS-CoV-2 and the N Protein
SARS-CoV-2 is a positive-sense, single-stranded RNA virus with a genome size of approximately 30,000 nucleotides. The virus's structural proteins include the Spike (S), Membrane (M), Envelope (E), and the Nucleocapsid (N) protein. The N-protein plays a pivotal role as it packages the viral RNA into a ribonucleoprotein complex and is crucial for viral replication and infection. Recent studies have linked N-protein to lung inflammation and acute lung injury. It has been observed that N-protein interacts with various cellular components, such as MASP-2 and NLRP3, leading to lung injury and inflammation.
 
RAGE: A Key Player in COVID-19
Receptor for Advanced Glycation Endproducts (RAGE) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. It is expressed predominantly in the lungs and is found on inflammatory and epithelial cells. RAGE is not only involved in binding with advanced glycation endproducts but also interacts with various ligands, including LPS, S100 proteins, and HMGB1. Recent studies have also shown that RAGE also plays a role in cancer development and cancer progression. Prior to this study, RAGE's potential role in COVID-19 pathogenesis had been proposed by various researchers, making it an intriguing target for further investigation.
 
The Groundbreaking Study
The study conducted at the Children's Hospital of Zhejiang University School of Medicine in Hangzhou, China, aimed to uncover the association between the SARS-CoV-2 N-protein and RAGE. By examining the interaction between these two key components, the researchers sought to understand their role in the acute lung injury induced by N-protein.
 
RAGE Identified as a Receptor for N-Protein
One of the primary findings of the study was the direct, high-affinity binding between the N-protein and RAGE. This binding was observed through various experiments, including flow cytometry-based binding assays, surface plasmon resonance analysis, and ELISA. The study indicated that RAGE possesses potent binding with the N-protein, similar to the binding affinity between the receptor binding domain of the SARS-CoV-2 S-protein and ACE2, which is essential for the virus's entry into host cells.
 
The researchers also discovered that the N-protein interacts with RAGE through both its N-terminal domain (NTD) and C-terminal domain (CTD). This dual-binding capability enhances the activation of the RAGE-mediated signaling pathway. HMGB1, another protein known to interact with RAGE, shares this feature of having two RAGE-binding domains.
 
N-Protein Activates the RAGE-ERK1/2-NF-ĸB Pathway
The study further delved into the signaling mechanisms triggered by the N-protein. In vitro experiments demonstrated that the N-protein activates the RAGE-ERK1/2-NF-ĸB signaling pathway, leading to a proinflammatory response. Notably, RAGE inhibition through antagonist treatment blocked N-protein-induced ERK1/2 activation, showing that RAGE acts upstream of ERK1/2 in this signaling cascade. This discovery highlights the significance of RAGE in mediating N-protein-induced inflammation.
 
N-NTD and N-CTD Mimic Full-Length N-Protein in Signaling and Inflammatory Response
The study also investigated whether the N-terminal domain (N-NTD) and C-terminal domain (N-CTD) of N-protein could replicate the signaling and inflammatory responses induced by full-length N-protein. Their experiments revealed that both N-NTD and N-CTD successfully triggered the RAGE-mediated signaling pathway and proinflammatory response, mirroring the effects of the full-length protein.
 
RAGE Deficiency Reduces N-Protein-Induced Inflammatory Response
To explore the role of RAGE in N-protein-induced inflammation, the researchers used bone marrow-derived macrophages (BMDMs) isolated from RAGE-deficient (RAGE−/−) and wild-type (WT) mice. Compared to WT BMDMs, RAGE-deficient BMDMs displayed reduced phosphorylation of ERK1/2 and NF-ĸB p65 in response to N-protein. Additionally, RAGE deficiency significantly diminished the effects of N-protein on the mRNA expression and cellular release of proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α. However, the study noted that RAGE deficiency did not completely eliminate the proinflammatory effects of N-protein, suggesting the involvement of other receptors or pathways.
 
RAGE Deficiency and Inhibition Alleviate N-Protein-Induced Acute Lung Injury
The investigation extended to in vivo experiments using mice. The researchers found that N-protein treatment elevated RAGE levels in the bronchoalveolar lavage (BAL) and lung. This increase in RAGE expression was accompanied by lung pathology and acute lung injury. Notably, RAGE deficiency in mice provided partial protection against N-protein-induced acute lung injury. Reduced lung injury in RAGE-deficient mice was evidenced by decreased edema, cellularity, protein concentration, inflammatory cell infiltration, and proinflammatory cytokines in the BAL.
 
Furthermore, the study explored the therapeutic potential of RAGE inhibition. By treating mice with a RAGE antagonist (RAP) in combination with N-protein, the researchers observed a significant reduction in lung pathology, total protein concentration, total cell count, neutrophil infiltration, and proinflammatory cytokines in the BAL. These results underscore the potential of targeting RAGE as a therapeutic strategy to mitigate acute lung injury induced by N-protein.
 
Implications and Future Directions
This groundbreaking study unravels a previously unrecognized aspect of SARS-CoV-2 pathogenesis by identifying RAGE as a receptor for the N-protein. This finding not only expands our understanding of the virus but also opens the door to potential therapeutic strategies that can mitigate COVID-19's most severe complications, such as acute lung injury and ARDS.
 
While these results are promising, several questions remain unanswered. Further research is needed to fully understand the interaction between RAGE and the N-protein and the role of RAGE in other aspects of SARS-CoV-2 infection. This discovery may also pave the way for the development of targeted therapies to combat COVID-19, ultimately providing new hope in the battle against the pandemic.
 
In Conclusion
The identification of RAGE as a receptor for the SARS-CoV-2 N-protein represents a remarkable breakthrough in COVID-19 research. This discovery has the potential to reshape our understanding of the virus's pathogenic mechanisms and offers novel avenues for therapeutic intervention. As researchers continue to delve deeper into these findings, the development of targeted therapies that can alleviate the severe acute lung injuries associated with COVID-19 may become a reality, ultimately bringing us one step closer to overcoming the pandemic.
 
The study findings were published in the peer reviewed American Journal of Respiratory Cell and Molecular Biology.
https://www.atsjournals.org/doi/full/10.1165/rcmb.2022-0351OC
 
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