A new study out of the Netherlands and published on the preprint server bioRxiv* in July 2020 discusses the commonly observed phenomenon of COVID-19 progression at 1-2 weeks from the onset of the earliest symptoms. This is ascribed to the overactivation of the inflammatory signaling cascade triggered by a hyperactive adaptive immune reaction, commonly called the cytokine storm.
Progressive Illness in COVID-19
The current pandemic of COVID-19 is primarily a severe pneumonic illness, but in most patients, it has a mild or asymptomatic phenotype. The severe form occurs in about one in five patients. It is characterized by bilateral pneumonia, quickly progressing to acute respiratory distress syndrome (ARDS), and death by respiratory failure, often complicated by multi-organ dysfunction.
The reason for such inappropriate immune stimulation is as yet unknown. The study shows that IgG antibodies to the spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The hyperactive immune system is characterized by high levels of cytokines that promote inflammation, including IL-6, IL-8, and TNF. On the other hand, early response antiviral cytokines like type I and III interferons (IFNs) are suppressed. The combination of low antiviral response with high pro-inflammatory cytokine levels causes a very unfavorable immune response in severe COVID-19.
Anti-spike IgG Induces High Inflammation
The severe lung injury seen in SARS first identified in 2003 has been shown earlier to be due to early high IgG production targeting the spike protein, which causes macrophages to be directed towards intense inflammation rather than tissue regeneration and repair. In this animal model, blocking Fc receptors could suppress inflammation and collateral damage. The same picture is observed in severely ill COVID-19 patients.
The present study aimed to examine the possibility that the anti-spike IgG antibodies are behind the excessive inflammation in these patients. They used macrophages induced to present the M2 phenotype and exposed them to spike-IgG a well as to the viral mimic PolyIC.
While individually, these stimuli produced low levels of IL-1β, IL-6, and TNF, and high levels of IL-8, vs. hardly any cytokine secretion, respectively, combined stimulation caused a steep increase in the secretion of IL-1β, IL-6, and TNF. These are associated with severe COVID-19. IL-10 secretion also surged though it is an anti-inflammatory cytokine. The researchers then confirmed the findings using primary lung macrophages.
SARS-CoV-2 viruses binding to ACE-2 receptors on a human cell, the initial stage of COVID-19 infection, conceptual 3D illustration. Image Credit: Kateryna Kon / Shutterstock
Anti-spike IgG Induces Pro-Inflammatory Changes in Macrophages
To further trace the cause of this inflammation, they looked at serum from 33 patients in the intensive care unit (ICU) who fell into one of three categories:
- No COVID-19
- COVID-19 positive but anti-spike IgG negative
- COVID-19 positive and anti-spike IgG positive
They found that as expected, the cytokines mentioned earlier were secreted at much higher levels in the third group. Follow-up RNA sequencing of macrophages stimulated with the serum from the patients in the third group also indicated that pro-inflammatory genes were being induced, but also IFN-β and IFN-γ. Further analysis showed that the enhanced macrophage secretion of these cytokines was associated with anti-spike IgG in a dose-dependent manner.
To sum up, the researchers say, “These data demonstrate that anti-Spike IgG immune complexes generated from the serum of severely ill COVID-19 patients induce a strong pro-inflammatory response by (otherwise immunosuppressive) human M2 macrophages, which is characterized by high production of classical cytokine storm mediators such as IL-1β, IL-6, IL-8, and TNF.”
Anti-Spike IgG Causes Endothelial Damage
Secondly, there is pulmonary edema due to the breakdown of the endothelium of the small blood vessel. Coagulation abnormalities are also found, including lung clots. They found that rather than exposure to PolyIC, which caused only a brief reduction in endothelial barrier strength, the combined stimulation of endothelium with spike protein and the serum from severe COVID-19 patients caused sustained disruption of the barrier, and increased adhesion of platelets to vascular endothelium.
Along with this, the endothelium released von Willebrand factor, which indicates procoagulability. Thus, the anti-spike IgG found in the serum of patients with severe COVID-19 causes not only macrophage-mediated hyperinflammation but enhanced damage to the microvascular endothelium and clots within the smallest blood vessels.
Glycosylation Pattern Stimulates Pro-Inflammatory Cytokines
Interestingly, they also found that the glycosylation pattern of the anti-spike IgG contributes to the strongly pro-inflammatory nature of these antibodies because the recombinant IgG fails to produce this level of stimulation. In fact, a reduction in the fucose content with an increase in galactose is a hallmark of anti-spike IgG1, compared to the sugar residue content of total IgG in the same patients.
The lower the fucose content, the higher was the production of IL-1β, IL-6, and TNF, but not IL-8 and IL-10. This low-fucose high-galactose pattern was then simulated in COVAI-18 to confirm that this was indeed the cause of the increased induction of pro-inflammatory cytokines.
The scientists concluded, “These data indicate that anti-Spike IgG from COVID-19 patients has an aberrant glycosylation pattern that makes these antibodies intrinsically more inflammatory than ‘common’ IgGs by increasing its capacity to induce high amounts of pro-inflammatory cytokines.”
Fostamatinib Inhibits Inflammation Induced by IgG
Next, they examined different means of counteracting the antibodies. They first found that these anti-SARS-CoV-2 IgG immune complexes bound to Fc gamma receptors (FcγRs), which are highly expressed on human macrophages. By selectively blocking each type of FcγR, they found that though all induced anti-spike IgG-mediated inflammation, this effect was most apparent with FcγRII. The low-fucose, high-galactose combination of sugars on the anti-spike IgG especially increases the affinity of the antibody for FcγRIII, but this is not the most prominent contributor to the hyperinflammatory response in severe COVID-19.
FcγRs are expressed by other cells, including human airway epithelium, which not only interacts closely with activated macrophages but presents a primary target for SARS-CoV-2.
The FcγR-signaling pathway depends on the kinase enzyme Syk, and blockade of this using R406, which is the active form of fostamatinib, a small molecule inhibitor, produced virtually complete inhibition of secretion of these cytokines induced by anti-spike IgG but not those induced by PolyIC stimulation alone.
At the gene level, they found this small molecule suppressed over 4,300 genes, and almost 4,000 genes were upregulated. Many of the suppressed genes encoded pro-inflammatory cytokines. Thus, “these data demonstrate that the excessive inflammatory response by anti-Spike IgG from severely ill COVID-19 patients can be counteracted by the Syk inhibitor fostamatinib.”
Fostamatinib may also inhibit the expression of Mucin-1 on epithelial cells, according to a recent study. This could mean that multiple pathways of inflammation are inhibited simultaneously.
Implications and Future Directions
Overall, therefore, the study contributes to explaining why severe COVID-19 disease often occurs around the time of activation of adaptive immunity. Other molecules involved in FcγR signaling could be targeted by other drugs as well. Again, FcγR stimulation induces human macrophages to undergo metabolic reprogramming. This could be yet another potential drug target.
These findings thus offer several clues towards the identification of therapies to suppress these hyperinflammatory phenomena and promote recovery from serious COVID-19 illness. Moreover, it may indicate that the aberrantly glycosylated IgGs found in the serum of convalescent COVID-19 patients who were severely ill should be filtered out before using the serum to treat other patients.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.