Since the beginning of the COVID-19 pandemic in late 2019, extensive research has been conducted on this disease and its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite significant progress, many aspects of this virus’s pathogenicity and mechanisms of action remain poorly understood (1). A notable case of a key question still unresolved is the molecular and pathogenic consequences of the interaction between the receptors that facilitate direct viral interaction with host cells and mediate viral entry (2, 3). Numerous studies have shown that human angiotensin-converting enzyme 2 (hACE2) acts as the primary receptor for SARS-CoV-2, binding to the viral spike protein (S) and allowing the virus to internalize into the host cell (4–8).

SARS-CoV-2 is a single-stranded, positive-sense RNA virus enclosed within a lipid envelope, and it encodes four structural proteins: envelope (E), membrane (M), nucleocapsid (N), S, the latter being the largest and most functionally significant (9, 10). From an immunological perspective, the S protein is the most immunogenic component of the virus. It assembles into trimers, forming large protrusions on the viral surface, which give the virus its characteristic “spike” appearance (11). The S protein consists of two distinct domains, S1 and S2, each with a specific role in viral entry. The S1 domain mediates initial interaction with receptor molecules on the host cell surface, while the S2 domain facilitates membrane fusion between the viral envelope and the host cell, enabling the release of the viral genome into the cytoplasm (12). Notably, within the S1 domain, the receptor-binding domain (RBD) exhibits high affinity for ACE2, binding strongly not only to the membrane-bound receptor but also to its soluble forms (2, 5, 7). Additionally, several host factors have been implicated in facilitating viral entry and membrane fusion. These include transmembrane proteases such as disintegrin and metalloprotease domain 17 (ADAM17), transmembrane protease serine 2 (TMPRSS2), and TNF-converting enzyme, as well as structural proteins like vimentin and clathrin, which may enhance viral binding and internalization (13–17).

An important consideration in the interaction between SARS-CoV-2 and ACE2 is the structural organization of the S protein. In its native state, S proteins assemble into trimers, forming club-shaped protrusions on the viral membrane surface. These trimeric structures are primarily responsible for host cell entry and membrane fusion (18). Spike trimers can adopt multiple conformational states, including the closed, open, and intermediate forms characterized by mobile but predominantly closed receptor-binding domains (RBDs). In the open conformation, the RBD of one or two monomers becomes exposed, allowing for ACE2 recognition and binding. Conversely, in the closed conformation, all three RBDs remain shielded by the N-terminal domain (NTD) of the S protein, limiting receptor accessibility (19, 20). Despite the established role of trimeric S in viral entry, it remains unclear whether monomeric S fragments, which may circulate in infected and vaccinated individuals, can elicit similar cellular effects and immune responses in the host (21–24). Therefore, further studies are needed to determine whether S monomers contribute to pathogenesis in vivo.

ACE2 functions both as an enzyme and as a critical receptor expressed on the surface of various cell types. It is most abundant in the gut, followed by the kidney, while its expression in the lung and heart is much lower. ACE2 can also be shed into the plasma, where it exists in two distinct forms: a membrane-bound (insoluble) form and a circulating soluble form, however the circulating ACE2 levels are very low to non-detectable, particularly in comparison to ACE (25–28). ACE2 plays a key role in the renin–angiotensin–aldosterone system (RAAS), a homeostatic regulator of vascular function (29). Within this system, renin, primarily produced by the kidneys, cleaves angiotensinogen derived from the liver to generate angiotensin I (Ang I). Subsequently, ACE converts Ang I into Ang II, which exerts its effects by binding to two distinct receptors: Ang II type 1 receptor (AT1R) and Ang II type 2 receptor (AT2R) (30, 31). Depending on the receptor engaged, Ang II can elicit divergent physiological responses. Binding to AT1R promotes vasoconstriction, oxidative stress, inflammation, and fibrosis, whereas activation of AT2R mediates opposing effects, including vasodilation and anti-inflammatory signaling (32, 33). Moreover, ACE2 counterbalances the activity of ACE by degrading Ang II into Ang-(1-7), a peptide with anti-inflammatory, vasodilatory and cardioprotective properties, thereby contributing to blood pressure regulation and cardiovascular homeostasis (34). SARS-CoV-2, particularly its RBD within the S1 subunit of the S protein, competes with Ang II for binding to hACE2 (6, 35). This interaction not only obstructs ACE2 activity but also downregulates its membrane expression, thereby disrupting the balance of the RAAS and contributing to disease pathogenesis (36–38). Interestingly, despite the high-affinity interaction between the viral RBD and ACE2, the catalytic active site of ACE2 remains unoccupied, and this binding occurs independently of the enzyme’s peptidase activity (39).

Since the onset of the COVID-19 pandemic, multiple SARS-CoV-2 variants of concern (VOCs) have emerged, each characterized by distinct mutations, primarily within the S protein (40). These genetic alterations have been associated with variations in disease severity, transmissibility, and immune evasion. The most recent VOC, designated Omicron (lineage B.1.1.529), was first detected in South Africa in November 2021 (41). Importantly, this variant has been reported to replicate approximately 70 times faster than the Delta variant; however, its capacity for deep lung tissue penetration appears to be reduced, correlating with a lower risk of severe disease and hospitalization. Genomic analyses have identified approximately 60 mutations distinguishing Omicron from the ancestral SARS-CoV-2 strain, with 32 of these mutations specifically affecting the S protein (42). Notably, several of these alterations had not been observed in previous VOCs (43). Among these mutations, Omicron exhibits 30 amino acid substitutions, three small deletions, and one small insertion within the S protein, with 15 of these changes localized to the RBD (44). Despite these extensive modifications, it remains unclear which specific mutations underline Omicron’s unique pathogenic and transmissibility characteristics. Further research is needed to determine their functional implications in viral entry, immune escape, and disease progression.

Our laboratory has already developed and validated a successful zebrafish model to study COVID-19 pathogenesis by injecting different variants of the S1 domain from the SARS-CoV-2 S protein into the hindbrain ventricle of zebrafish larva (45–47). In this approach, the larval hindbrain ventricle mimics the microenvironment of human pulmonary alveoli (48), enabling the study of local and systemic immune responses, including COVID-19-associated cytokine storm syndrome (CSS), emergency hematopoiesis, and hemorrhagic events (45, 46). Zebrafish possesses a single copy of the ace2 gene in its genome, which is highly conserved in both sequence and structure compared to its mammalian counterpart (49). Recent single-cell RNA sequencing analyses have further demonstrated that the RAAS signaling pathway is also evolutionarily conserved between humans and zebrafish (50). In the present study, the zebrafish model has been used to elucidate the dual effect of ACE2 in the pathogenesis of COVID-19-associated CSS using monomeric and trimeric forms of wild-type S1 (S1WT) and to explore the inflammatory properties of Omicron S1. Our findings provide insights into the immunopathogenic mechanisms of SARS-CoV-2 and its emerging variants.

The Omicron variant from SARS-CoV-2 is characterized by numerous mutations in its spike protein, particularly in the receptor-binding domain, leading to increased transmissibility and reduced antibody and vaccine effectiveness. These changes result in distinct biological behavior compared to earlier variants, including altered immune responses and pathogenicity. Studying different variants individually is essential, as each may differ in how it spreads, evades immunity, and triggers inflammation. Understanding these differences is key for developing effective diagnostics, treatments, and public health strategies (56, 57). Our in vivo findings indicate that the Omicron variant of SARS-CoV-2 elicits a distinct immune response compared to ancestral variants. In contrast to the S1WT protein, S1 Omicron induced significantly lower recruitment and expansion of neutrophils and macrophages at all examined time points. Despite this attenuated cellular response, Omicron triggered a more pronounced systemic inflammatory response, characterized by elevated Nfkb activity and increased expression of selected pro-inflammatory cytokines, particularly in the inflammatory site. Notably, S1 Omicron induced stronger caspase-1 activation both locally and systemically than S1WT, suggesting variant-specific activation of the inflammasome. These results imply that S1 Omicron may promote inflammation via alternative immune pathways, with a prominent role for inflammasome activation. This observation is supported by in vitro experiments in human monocytic THP-1 cells, where Omicron variant infection led to the highest IL-1β secretion (58). Enhanced inflammasome activity may contribute to more efficient viral neutralization and could partially account for the observed attenuation in clinical severity (58).

Emerging evidence suggests that the Omicron variant modulates neutrophil cell death through mechanisms distinct from those of earlier SARS-CoV-2 variants. Neutrophils are essential components of the innate immune system, and their activation followed by cell death via apoptosis, pyroptosis, or NETosis, plays a pivotal role in regulating inflammation and mediating pathogen clearance (59–61). Despite the generally milder respiratory manifestations associated with Omicron infections, our findings demonstrate that this variant induced substantial neutrophil cytotoxicity. This increased cell death, likely driven by enhanced inflammasome activation, may contribute to the lower virulence observed with Omicron compared to the wild-type strain and other variants of concern. Notably, our data also shows similar kinetics in macrophage dynamics, supporting the hypothesis of a shared inflammasome-dependent mechanism. These observations are consistent with previous reports indicating that Omicron can induce inflammasome-mediated death in both neutrophils and macrophages (51–53, 55, 62). Further studies are warranted to elucidate the molecular pathways through which Omicron influences myeloid cell survival and death, which could uncover novel therapeutic targets for modulating host responses in SARS-CoV-2 infection.

Although previous in vitro studies have demonstrated the high interaction and affinity between the SARS-CoV-2 S protein and the human ACE2 receptor, unfortunately a more comprehensive understanding of the dynamic immune response, the receptor engagement, and the downstream signaling pathways activated following such interaction cannot be fully captured in cell culture systems (63, 64). The zebrafish model offers a unique opportunity to dissect the complex interactions between viral proteins and host receptors in vivo, shedding light on the role of ACE2 in modulating the immune response and providing insights into the broader implications of ACE2-mediated signaling in COVID-19 pathogenesis (45, 46, 65). In fact, we have previously reported that the zebrafish Ace2 plays a conserved anti-inflammatory role in modulating COVID-19-associated CSS via the production of Ang-(1-7) (45). We confirm in the present study the evolutionary conservation of this axis by demonstrating that overexpression of human ACE2 in zebrafish larvae resulted in accelerated resolution of the inflammation upon S1WT injection, likely through the production of Ang-(1-7), since endogenous zebrafish Ace2 failed to neutralize monomeric S1WT. Specifically, one of the most interesting results of our study is that the trimeric S1/S2WT hardly induced the recruitment of neutrophils and macrophages at the injection site and failed to promote a systemic inflammatory response, while genetic inhibition of zebrafish Ace2 fully restored its hyperinflammatory activity. This is consistent with the 100-fold lower affinity of monomeric S1 compared with trimer S1/S2 (EC₅₀ of 300–700 and 4–24 ng/mL, respectively) (https://www.sinobiological.com/recombinant-proteins/2019-ncov-cov-spike-40591-v08b1, https://www.sinobiological.com/recombinant-proteins/sars-cov-2-cov-spike-40589-v08h8). The attenuated phagocyte recruitment and diminished inflammatory gene expression observed in response to the trimeric form are consistent with patterns described in other viral infections, where the structural integrity of viral proteins critically influences immune activation and may facilitate immune evasion mechanisms (20, 66, 67).

Emphasizing the importance of our data, although the SARS-CoV-2 spike protein is initially present in a trimeric conformation on the viral surface, it undergoes proteolytic fragmentation during infection, and monomeric spike fragments have been detected in the bloodstream of infected patients, persisting even several months after recovery (22–24). Moreover, recombinant vaccines comprising residues 319–545 of the RBD of the S protein have also been used to elicit robust functional antibody responses in mice, rabbits, and non-human primates (21). Furthermore, this also aligns with other previously published results revealing that trimeric S1 serves as a potent inhibitor of SARS-CoV-2 infection, exhibiting affinity for ACE2 and effectively blocking viral entry more efficiently than monomeric RBD (68). These results confirm other anti-inflammatory role for ACE2 in direct neutralizing of the trimeric fragments of Spike leading to diminish of the inflammatory effects that are seen with monomeric forms of S protein. Therefore, all these findings together emphasize the complexity of the host-pathogen interactions in viral infections and provide potential targets for future therapeutic strategies for COVID-19, including the design of vaccines and the development of novel strategies to prevent SARS-CoV-2 infection from spreading to neighboring cells and, therefore, to control viral dissemination within the host.

In summary, our study reveals that the S1 domain of the Omicron variant triggers a more robust proinflammatory response than the ancestral S1WT, primarily through enhanced inflammasome activation and promotion of neutrophil and macrophage death. We further demonstrate that both the trimeric full-length Spike protein and its monomeric fragments—generated during infection—contribute to the COVID-19-associated CSS, likely via TLR2 and inflammasome activation (Figure 9). Importantly, we identify a dual anti-inflammatory role for ACE2 in SARS-CoV-2 pathogenesis: (1) enzymatic production of Ang-(1–7), and (2) direct neutralization of trimeric Spike, thereby preventing immune overactivation. These findings highlight the differential immunostimulatory potential of Spike-derived fragments and underscore the importance of dissecting their interactions with host immune pathways. A deeper understanding of these dynamics is essential to clarify their roles in both acute disease and post-acute sequelae (long COVID) and may inform the development of targeted therapeutic strategies aimed at modulating inflammasome activity and ACE2-mediated regulation.