A novel role for macrophage-activation signaling in orchestrating the recruitment process in response to the SARS-CoV-2 spike protein

By Pooja Toshniwal PahariaReviewed by Danielle Ellis, B.Sc.May 19 2023

In a recent study published in Scientific Reports, researchers derived peptides from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein to assess coronavirus disease 2019 (COVID-19)-associated inflammatory changes using zebrafish models.

Background

The COVID-19 pandemic continues to affect millions of humans and overburden healthcare facilities across the globe despite vaccination and other mitigation efforts. The continual emergence of novel SARS-CoV-2 variants of concern (VOCs) has threatened the efficacy of existing therapies, warranting the development of novel agents.

Improving our understanding of COVID-19 pathophysiology could aid in developing more effective agents to improve the standard of care and reduce the global burden of COVID-19. Manipulating the entire virus requires heightened biosafety levels, and therefore, alternate strategies, including peptide synthesis from SARS-CoV-2 proteins, could provide a more feasible and swifter solution.

About the study

In the present study, researchers investigated whether the peptide synthesis approach and zebrafish models could be used to elucidate COVID-19 pathophysiology.

The team performed an in-silico analysis to synthesize two peptides, PSPD2002 and PSPD2003, from SARS-CoV-2 spike (S) protein, and they were validated in vitro as well as in vivo. The peptides were quantified using anti-S immunoglobulin G (IgG). Fluorescence-activated cell sorting (FACS) was performed to obtain neutrophils and macrophages and assess their activation and inflammatory responses to the peptide challenge in vitro.

Subsequently, the peptides were injected into transgenic zebrafish larvae to assess macrophage polarization, survival, and oxidative stress biomarkers after six days of fertilization. The SARS-CoV-2 spike protein-derived peptides were purified using high-performance liquid chromatography (HPLC). Human angiotensin-converting enzyme 2 (ACE2) from the Saccharomyces cerevisiae strain was expressed in BY4742 yeast to evaluate the effects of the peptides on ACE2 activity.

Further, molecular dynamic (MD) simulations were performed. Confocal microscopy (CFM) was used to evaluate COVID-19-associated inflammation, and zebrafish splenic, hepatic, intestinal, and muscle tissues were obtained for histopathological examination. Oxidative stress levels were measured and cytotoxicity assays were performed.

Oxidative stress markers included nitric oxide (NO), thiobarbituric acid reactive species (TBARS), reactive oxygen species (ROS), and hydrogen peroxide (H₂O₂). Protein levels were measured using enzyme-linked immunosorbent assays (ELISA). Flow cytometry was performed to assess membrane-bound L-selectin (CD62L)-based neutrophil activation, and murine alveolar macrophage AMJ2-C11 cells were used to assess cytotoxicity.

Results

The peptides were derived from areas of robust S-ACE2 interaction. The in-silico assays and MD simulations showed that the S protein-based peptide molecules were bound to ACE-2 receptors stably and interacted with adhesion molecules and other receptors from zebrafish and humans, including the T-cell receptor (TCR) and major histocompatibility complex (MHC).

PSPD2002 showed binding with ACE2 receptors in zebrafish and humans, with values of −7.4 and -8.0 kcal per mol, respectively. The corresponding affinity values for PSPD2003 were -8.2 and -8.1 kcal per mol, respectively. Peptide-stimulated macrophages demonstrated the increased generation of nitric oxide, chemokine C-X-C motif ligand-2 (CXCL-2), and tumor necrosis factor-alpha (TNF-α).

Peptide inoculation in zebrafish larvae stimulated inflammatory processes characterized by the recruitment of macrophages, histopathological alterations, and enhanced mortality, comparable to changes observed among COVID-19 patients. Both peptides could be quantified in vitro and were detected by anti-S IgG. They demonstrated strong antigenic potential and interacted with zebrafish and human immunological receptors. Therefore, the peptides could be used for developing COVID-19 vaccines and other therapeutics.

PSPD2003 demonstrated dose-dependent nitric oxide production, and PSPD2003 induced CXCL2 and TNF-α production. Neutrophils were stimulated by PSPD2002 and PSPD2003 at 10.0 and 100.0 μg/ml, respectively; however, the peptides could not modulate CD62L activity on the cellular surface, irrespective of lipopolysaccharide (LPS) presence or absence. The findings indicated that the peptides produced macrophage-mediated pro-inflammatory responses.

PSPD2002 (10.0 μg/ml) and PSPD2003 (1.0 and 10.0 μg/ml) lowered the survival rate among animals, and PSPD2003 induced more intense inflammation than PSPD2002, indicating that PSPD2003 was more cytotoxic than PSPD2002. Higher peptide concentrations induced inflammation more rapidly, peaking at two days post-immunization, followed by quicker resolution. The peptide injections decreased H₂O₂ and superoxide dismutase (SOD) levels but increased malondialdehyde (MDA) production. PSPD2002 decreased nitrite levels, whereas PSPD2003 increased catalase (CAT) levels.

The extent of immunological infiltration correlated with the redox profiles of the zebrafish larvae. Docking analysis findings showed that the affinity values for all peptide-antioxidant interactions negatively surpassed the threshold of −6.00 kcal per mol. For PSPD2022 interactions with CAT and SOD, the team obtained affinity values of -6.70 and −7.20 kcal per mol, respectively. Concerning PSPD2003, affinity values for the corresponding enzymes were -7.20 and −6.60 kcal per mol, respectively.

Conclusion

Overall, the study findings showed that peptide synthesis could be a valuable approach to evaluate SARS-CoV-2-associated inflammation in the host. In addition, zebrafish may be used in animal studies to simulate COVID-19-associated inflammation in humans.