Targeting C5a/C5aR1 signaling improves COVID-19 immunopathology

A recent study posted to the bioRxiv* preprint server investigated the role of complement component 5a (C5a) and its receptor (C5a receptor type 1, C5aR1) in the pathophysiology of coronavirus disease 2019 (COVID-19).

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS), leading to organ dysfunction and death. Studies have shown at least partial effectiveness of anti-inflammatory drugs in managing the severity of COVID-19. However, these therapies may affect the host’s immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other secondary infections.

The development of new drugs targeting the inflammatory response should be centered around the mediator, which is essential for immunopathology but not necessary for infection control. As such, C5a/C5aR1 signaling could be a possible candidate. C5aR1 signaling has been implicated in several inflammatory diseases. In addition, mounting evidence points to the potential role of the complement system in the pathophysiology of COVID-19.

The study and findings

In the current study, researchers investigated the role of C5a/C5aR1 signaling in COVID-19. They analyzed the C5a levels in bronchoalveolar lavage (BAL) fluid of critical COVID-19 patients requiring mechanical ventilation and compared them to mechanically ventilated flu patients. C5a levels were higher in COVID-19 patients than in influenza patients, indicating that C5a was activated locally in COVID-19 patients and may have activated C5aR1 signaling.

The authors examined a database of single-cell transcriptomes to identify cells that express C5AR1 in the BAL fluid of COVID-19 and non-COVID-19 pneumonia patients. C5AR1 expression was higher in neutrophils, which are abundant in BAL fluid from COVID-19 patients compared to non-COVID-19 pneumonia patients. It was also expressed in monocytes/macrophages in patients from both groups. The single-cell transcriptomic data was validated by immunostaining of C5aR1 from the lungs of observed COVID-19 patients with simultaneous staining of neutrophils and monocytes/macrophages.

Consistent with the transcriptome data, C5aR1 expression was enriched in neutrophils and monocytes/macrophages. Next, the team assessed the correlation of C5a levels with several inflammatory cells/markers known to be enriched in BAL fluid from COVID-19 patients. C5a levels correlated with degranulating/hyperactivated neutrophils and CXC motif chemokine ligand 8 (CXCL8).

The researchers also examined SARS-CoV-2-infected K18-hACE2 transgenic (Tg) mice. Elevated levels of C5a were observed in the lungs of infected mice. In addition, the clinical signs (weight loss and clinical score) and COVID-19 pathology of mice were associated with elevated levels of pro-inflammatory chemokines/cytokines in the lungs. Immunofluorescence analyzes with Tg^Phlox/phlox mice (with enhanced green fluorescent protein reporter) revealed that C5aR1 was expressed mainly in neutrophils and macrophages.

The team created a group of transgenic mice lacking C5aR1 signaling (Tg^cKO mice) and repeated the studies. Histopathological examination revealed less severe damage to lung tissue in the Tg^cKO mice. This was associated with a decrease in pro-inflammatory chemokines/cytokines. Nevertheless, there were no visible differences in viral load between Tg^cKO and Tg^phlox/phlox mice, indicating that the C5aR1 signaling was involved in lung pathology in COVID-19 without playing a role in controlling viral infection. Given the apparent involvement of C5a/C5aR1 signaling in COVID-19 immunopathology, the efficacy of an orally acting allosteric antagonist of C5aR1, DF2593A, was evaluated in the mice.

Mice were treated with the inhibitor one hour before infection with SARS-CoV-2 and once daily for five days. They noted that drug treatment improved the clinical signs of infected mice compared to vehicle-treated mice. It also improved lung pathology in infected mice, although the viral load was unchanged. Moreover, in vitro analysis also showed that the compound was an ineffective inhibitor of SARS-CoV-2 replication. The decrease in lung pathology was associated with lower levels of pro-inflammatory chemokines/cytokines.

Furthermore, they assessed whether the absence of C5aR1 signaling in myeloid cells would affect their infiltration into the lung tissue of infected mice. The infiltration of total leukocytes, myeloid cells, neutrophils and inflammatory monocytes was similar in the lungs of the Tg^cKO and Tg^Phlox/phlox mice infected with SARS-CoV-2. The infiltration of myeloid cells, inflammatory monocytes, or neutrophils was not affected by DF2593A treatment, although total leukocyte infiltration was reduced.

The team tested whether C5aR1 signaling was involved in the formation of neutrophil extracellular traps (NETs) in infected mice. The levels of NETs were significantly lower in Tg^cKO mice than Tg^phlox/phlox mice. In line with this, human neutrophils are infected in vitro SARS-CoV-2 generated higher levels of NETs in the presence of small amounts of recombinant C5a.

conclusions

These findings confirm the possibility of using a C5aR1 signaling inhibitor for the treatment of COVID-19. In particular, the hypothesis that inhibiting this signaling pathway would be beneficial in COVID-19 may raise concerns about the secondary infections commonly seen in COVID-19 patients. Overall, the study clarified the critical role of C5aR1 signaling for lung immunopathology and showed that inhibition of the signaling pathway could be an alternative treatment for COVID-19.

*Important announcement

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, should guide clinical practice/health-related behavior, or be treated as established information.