Complement pathways as potential targets
The available data suggest that the LP and the CP are the two major complement pathways activated in response to SARS-CoV-2. MBL, collectin 10 and -11 as well as the ficolins 1-3 function as soluble sensors of the LP (Garred et al., 2016). They interact with the serine protases MASP1 and MASP2 that act in concert to cleave C4 and C2 and form the C3 convertase C4b2b which proteolytically cleave C3 into C3a and C3b (Figure 1). MBL comprises multiple carbohydrate recognition domains that can bind to the high-mannose structure of the SARS-CoV S protein (Ip et al., 2005). In particular, the N-linked glycosylation site, N330, on the S protein seems to be critical for MBL interaction. Further, MBL directly inhibited SARS-CoV-mediated infection in vitro (Zhou et al., 2010). In addition to the S protein, a recent report uncovered the interaction of LP MASP-2 with a highly conserved motif in the N protein of SARS-CoV, SARS-CoV-2 and MERS-CoV. Intriguingly, this interaction not only potentiated MASP-2-driven activation of the LP but aggravated LPS-induced pneumonia in a MASP-2-dependent way (Gao et al., 2020). Given that the N-protein is one of the most abundant structural proteins in the serum of patients infected by SARS-CoV (Che et al., 2004; Chen et al., 2005; Guan, Chen, Foo, Tan, Goh & Wee, 2004) these data indicate that the N protein-mediated interaction with MASP-2 could serve as an important amplifier of LP activation in highly pathogenic coronavirus infection. Thus, the available data point toward the LP as an important target in these virus infections.
C1q is the sensor molecule of the CP that recognizes multiple conserved molecular patterns including IgM or IgG hexamer molecules that have bound to their cognate antigen. Similar to MASP-1 and MASP-2, the serine proteases C1r and C1s form a complex with IgG/IgM-bound C1q to cleave C4 and C2 and generate the C4b2b convertase eventually cleaving C3 into C3a and C3b (Figure 1). Within the first week after symptoms, COVID-19 infection results in the production of S protein-specific IgG/IgM antibodies as a target structure for C1q (Wolfel et al., 2020). A recent study from Wuhan shows that during the first 5 days after clinical onset, already 30-40% of the infected individuals have generated IgM or IgG antibodies directed against the S or N proteins of SARS-CoV-2, with a slightly higher frequency of antibodies against the S protein (Liu et al., 2020). After 5 weeks, all of the 214 tested patients showed IgG seroconversion. These data suggest that the seroconversion is somewhat quicker than what has been observed with SARS-CoV (Peiris et al., 2003). Taken together, CP activation by IgM and IgG antibodies directed against the S and N proteins of SARS-CoV and SARS-CoV-2 serves as second mechanism of complement activation in addition to the initial virus sensing by the MBL and LP activation (Figure 1).
The C3 convertase C4b2b, which is assembled in response to LP and CP activation generates the AT C3a from C3, which can degranulate basophils and mast cells leading to histamine release through activation of its cognate C3aR (el-Lati, Dahinden & Church, 1994; Kretzschmar et al., 1993). C3aR expression is triggered in neutrophils upon LPS exposure and contributes to NETosis (Guglietta et al., 2016). Further, C3a induces aggregation and serotonin release from platelets, regulates secretion of IL-6 and TNF-α from B-cells and monocytes and leads to the production of IL-8 by an epithelial cell line (Fischer & Hugli, 1997; Fischer, Jagels & Hugli, 1999; Fukuoka & Hugli, 1988). Intracellularly, C3a plays an important role in activating the NLRP3-inflammasome in human monocytes (Asgari et al., 2013). Taken together, C3a can mediate a strong pro-inflammatory environment. The C3 convertase also serves as the nucleus of the C5 convertase when C3b molecules form a complex with C4b2b resulting in C4b2b3b, the C5 convertase of the LP and the CP which cleaves C5 into the AT C5a and C5b (Figure 1) (Ekdahl et al., 2019). Similar to C3a and in concert with C3a, C5a can drive a strong pro-inflammatory environment through its strong chemotactic properties on neutrophils, monocytes, eosinophils, basophils, mast cell and dendritic cells (DC) and it strong potency to activate such cells to release ROS, lysosomal enzymes as well as pro-inflammatory cytokines such as IL-1β, TNF-α, IL-6 and chemokines of the CC and CXC families (Figure 2). C5a also drives the activation and differentiation of T cells through DC maturation downstream of C5aR1 (Antoniou et al., 2020; Weaver et al., 2010). Also, C5a controls histamine-induced increase in vasopermeability (Kordowski et al., 2019) and drives the production of metabolites of the arachidonic acid lipoxgenase and cyclooxygenase pathways resulting in increased production of leukotriene B4 (LTB4) or prostaglandine E2, both of which increase vasopermeability (Karasu, Nilsson, Kohl, Lambris & Huber-Lang, 2019; Klos, Tenner, Johswich, Ager, Reis & Kohl, 2009).
Importantly, such increased vasopermeability of the alveolar-capillary interface, massive neutrophil recruitment associated with tremendous production of proinflammatory cytokines and chemokines including IL-1β, IL-6, IFN-γ, IL-8, CXCL10 and CCL2 leading to the development of ALI/ARDS has been observed in infections with highly pathogenic respiratory viruses including H5N1 influenza, SARS-CoV and MERS-CoV (Jiang et al., 2019; Jiang et al., 2018). In experimental models as well as in patients infected with influenza (Ohta, Torii, Imai, Kimura, Okada & Ito, 2011), SARS-CoV (Gralinski et al., 2018), MERS-CoV (Jiang et al., 2018) or SARS-CoV-2 (Gao et al., 2020; Magro et al., 2020) increased blood levels and/or lung deposits of complement activation products have been described.
Taken together, the picture emerges that highly pathogenic coronaviruses activate complement by the LP and CP. This activation drives the generation of huge amounts of the highly proinflammatory cleavage products C3a and C5a, when complement activation is not sufficiently controlled by complement regulator proteins (Figure 3). Further, TMA results in C3 and C5 cleavage of by non-canonical pathways through serine proteases located in the intracellular space of in the vasculature that exert promiscuous enzyme activity (Figure 2) (Ekdahl et al., 2016). Below, we will discuss strategies to prevent the initiation of LP and CP, to attenuate convertase-mediated amplification and to inhibit the effector functions of C5a/C5aR1 axis activation.