Scientists crack the truth of Covid-19’s death. Immune cells ＂frustrated infection＂ induced inflammatory storm

The COVID-19 epidemic has lasted for more than four years, and Covid-19 is still raging around the world. According to statistics, nearly 7 million people infected with COVID-19 around the world have died of infection. In fact, the main cause of illness deterioration and even death of most patients is not the virus itself, but the excessive inflammatory reaction and inflammatory factor storm caused by Covid-19. However, the specific mechanism of how the virus triggers such a strong inflammatory response in the lungs is still unclear.

Recently, the Chinese research team of the University of Southern California published an important achievement in the journal Nature Cell Biology. Studies have revealed that Covid-19 can trigger a stronger inflammatory response than other RNA viruses. Surprisingly, this inflammatory factor storm is not caused by the rapid replication infection of the virus to lung epithelial cells, but by the "frustration infection" of the virus to a large number of immune cells pouring into the lungs in the later stage, that is, the virus can not complete the replication infection process.

The researchers further revealed the mechanism of Covid-19 infecting immune cells and triggering the storm of inflammatory factors: Toll-like receptor 1(TLR1) on the surface of immune cells recognizes the structural proteins E (envelope glycoprotein) and M (matrix protein) of Covid-19, so that the virus is endocytosed, thus causing "frustration infection". In addition, the binding of Covid-19 envelope glycoprotein E to immune cells TLR1 can directly activate the inflammatory response of immune cells, leading to the release of pro-inflammatory factors, which is the root cause of cytokine storm caused by Covid-19.

Dr. Duan Tianhao, Dr. Xing Changsheng and Dr. Chu Junjun are the co-first authors of this paper, and Professor Wang Rongfu is the correspondent author.

In order to explore the uniqueness of Covid-19’s strong inflammatory response, scientists screened 27 kinds of protein coded by Covid-19. Studies have revealed that Covid-19-encoded protein named NSP14 can directly promote the phosphorylation of IKK, a key kinase complex in the inflammatory signaling pathway, and significantly enhance the inflammatory response.

Interestingly, another Covid-19-encoded protein named ORF6 can inhibit inflammatory reaction and can be anchored to cell membrane. ORF6 significantly inhibits the activation of inflammatory reaction by preventing the nuclear entry of inflammatory transcription factor NF-κ B..

However, NSP14 promotes inflammation and ORF6 inhibits inflammation. What will be the final result of these two actions? Scientists have noticed that there was no obvious inflammatory reaction in the early stage of Covid-19 infection, but the inflammatory reaction increased significantly in the middle and late stage. This suggests that NSP14 and ORF6 may have different expression patterns at different stages of infection, which may lead to clinical observation.

After consulting the data, scientists noticed that there was a large amount of immune cell infiltration in Covid-19’s lungs at the late stage of infection. This leads to a guess: Will these infiltrated immune cells be infected by Covid-19, and then release a large number of inflammatory factors?

To answer these questions, researchers conducted a series of experiments. They used a modified version of Covid-19 labeled with green fluorescence to infect lung epithelial cells and immune cells respectively. The results showed that only lung epithelial cells showed fluorescent signal, indicating that Covid-19 could only replicate when infected with epithelial cells, but could not replicate when infected with immune cells (frustration infection). On the contrary, although a low level of inflammatory reaction activation was detected in lung epithelial cells, a strong inflammatory signal activation was detected in immune cells.

Scientists speculate that the different expression patterns of NSP14 and ORF6 in lung epithelial cells and immune cells may lead to the inhibition of inflammatory reaction in epithelial cells and the activation of inflammatory reaction in immune cells, which may be related to Covid-19’s "frustrated infection" in immune cells.

Traditionally, lung epithelial cells are the main target of infection in Covid-19, because there is a key receptor protein ACE2 on its surface, which can bind to Covid-19 spike protein S and promote the virus to invade cells. Immune cells, on the other hand, are not considered to have ACE2 receptors, so they are usually not considered to be infected by Covid-19.

However, further research shows that Covid-19 can infect immune cells despite the lack of ACE2 receptor, but due to the lack of ACE2, the virus cannot replicate after entering immune cells (frustration infection). What is even more surprising is that this "frustration infection" causes Covid-19 to selectively express NSP14 protein which promotes inflammatory response, but not ORF6 protein which inhibits inflammation, thus aggravating the inflammatory response of immune cells and promoting the generation of inflammatory storm.

Accordingly, scientists describe the complete mechanism of inflammation induced by Covid-19:

In the early stage, Covid-19 mainly infected lung epithelial cells through ACE2 receptor, where the virus quickly replicated and released a large number of virus particles to infect other epithelial cells. Although lung epithelial cells also express a small amount of TLR1 receptor and can be activated by virus, the expression of TLR1 in epithelial cells is much lower than that in immune cells, and the activation level of inflammatory signals is weak. In addition, due to Covid-19’s massive expression of inflammatory inhibitory protein ORF6 in epithelial cells, the inflammatory reaction was effectively inhibited. Therefore, the early stage of Covid-19 infection is mainly characterized by virus replication, accompanied by weak inflammatory reaction, that is, asymptomatic infection period.

In the late stage, the viral load reached the peak, the replication speed decreased, and a large number of immune cells gathered in the lungs. Immune cells recognize and bind the structural proteins E and M of the virus through the highly expressed TLR1 receptor, which triggers the activation of severe inflammatory reaction. At the same time, the virus binding to TLR1 can enter the immune cells through endocytosis. This "frustrated infection" mode prevents the virus from forming a replication and transcription complex RTC to transcribe the subgenomic RNA of the virus. Due to the lack of subgenomic RNA, viral structural proteins and viral accessory proteins including ORF6 cannot be expressed, but the pro-inflammatory protein NSP14 can be directly translated through genomic RNA. Because only NSP14 is expressed without ORF6, the inflammatory reaction is further aggravated, and immune cells release a large number of inflammatory factors, triggering a storm of inflammatory factors.

In addition, scientists have also studied other human coronaviruses of Coronaviridae. They found that SARS-CoV-1 triggered a large number of inflammatory reactions through the same molecular mechanism as SARS-CoV-2, and even the ability of its E protein to induce inflammation in immune cells was higher than that of SARS-CoV-2. This explains why the mortality rate of SARS-CoV-1 was as high as 11% when it broke out 20 years ago, far exceeding the current epidemic of SARS-CoV-2.

In contrast, other mild human coronaviruses, such as OC43, NL63 and 229E, have weak binding ability to TLR1, which also explains that their symptoms are usually mild.

This study reveals in detail the complete molecular mechanism of Covid-19 from its early entry into the human body for replication to its late stage, which leads to severe inflammation and death of patients, and provides important theoretical guidance for the treatment of severe COVID-19 patients. At the same time, the study also found that the small molecule inhibitor of TLR1 may be a better choice for the treatment of severe COVID-19 infection.