Targeting the Complement–Sphingolipid System in COVID-19 and Gaucher Diseases: Evidence for a New Treatment Strategy

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2)-induced disease (COVID-19) and Gaucher disease (GD) exhibit upregulation of complement 5a (C5a) and its C5aR1 receptor, and excess synthesis of glycosphingolipids that lead to increased infiltration and activation of innate and adaptive immune cells, resulting in massive generation of pro-inflammatory cytokines, chemokines and growth factors. This C5a–C5aR1–glycosphingolipid pathway- induced pro-inflammatory environment causes the tissue damage in COVID-19 and GD. Strikingly, pharmaceutically targeting the C5a–C5aR1 axis or the glycosphingolipid synthesis pathway led to a reduction in glycosphingolipid synthesis and innate and adaptive immune inflammation, and protection from the tissue destruction in both COVID-19 and GD. These results reveal a common involvement of the complement and glycosphingolipid systems driving immune inflammation and tissue damage in COVID-19 and GD, respectively. It is therefore expected that combined targeting of the complement and sphingolipid pathways could ameliorate the tissue destruction, organ failure, and death in patients at high-risk of developing severe cases of COVID-19.


COVID-19 Vaccines and Alternative Treatments
Several vaccines have been developed against multiple strains of SARS-CoV-2 infection, including mRNA vaccines, viral vector vaccines, inactivated vaccines, and proteinbased vaccines [130] (Table 5). mRNA vaccines have shown an efficacy ranging from 48 to >90% at 5-6 months' follow-up post second vaccine dose [130]. Viral vector vaccines had an efficacy ranging from 65 to 91.6% (Sputnik), around 70% (in Brazil), or between 10 and 64% (South Africa), according to the different vaccine types and countries in which they were used [130]. Finally, inactivated and subunit vaccines, showed an efficacy ranging from 50 to 91.6%, again based on the different subtypes and the country in which they were employed [130]. SARS-CoV-2 continuously develops mutations (variants) and will continue to do so because relatively few people globally have been vaccinated [131]. These findings suggest that the variability in vaccine efficacy is linked to the dominant variant circulating in the country at the time.
Despite the high efficacy of different vaccines, multiple studies have shown the phenomenon of immunity waning occurring approximately 6 months after vaccine administration [132]. In addition, all of the vaccines can be associated with severe adverse events, namely severe anaphylaxis, myocarditis/pericarditis, lymphadenopathy, appendicitis, herpes-zoster infection, viral facial palsy, transverse myelitis, Guillain-Barré syndrome, encephalopathy, thromboembolism, and thrombosis-thrombocytopenia syndrome [130]. These severe adverse events are rare, and may vary significantly according to specific features of the target population such as age. For example, myocarditis is more frequently reported in males under the age of 30 when compared to other population categories [133].
Several drugs, mainly belonging to the classes of antivirals, antibiotics, immunemodulators, immune-suppressants, or anti-inflammatory molecules, have also been proposed for treating COVID-19 and its complications ( Table 5). Some of these drugs have been used off-label and their efficacy is still debated. Lopinavir and ritonavir are two protease inhibitors used for the treatment of human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) [134,135]. Their use in combination has been proposed for COVID-19 [135,136]. These drugs may also prevent the secondary immune burst without causing severe adverse events at early stages of COVID-19 and are therefore not recommended for COVID-19 at severe stages of the disease [137]. Among several repurposed drugs currently used in patients with COVID-19, the nucleoside analogs are a preferred class [138], with two drugs in particular: (1) the nucleotide precursor favipiravir, and (2) the nucleoside analogue remdesivir, which inhibit the RDRP [139,140]. A recent systematic review of the literature has shown that favipiravir may significantly improve the clinical condition of COVID-19 patients, although the results suggested no significant differences in the length of hospitalization and clinical recovery [138]. A combination of favipiravir with other therapies (e.g., tocilizumab) has shown more promising results in improving a patients' clinical status [138]. Possible safety concerns for favipiravir include increased blood uric acid and teratogenicity, as the medication is secreted in semen and breast milk and has demonstrated toxicity in animal studies [141]. Remdesivir may help improve recovery and the clinical outcomes of hospitalized COVID-19 patients, although its effects in reducing mortality are still uncertain [142,143]. It should be administered in a hospital setting. Unfortunately, data on its possible toxicity are sparse [138,144].

SARS-CoV-2 Reinfection
Several cases of SARS-CoV-2 reinfection after vaccination/first infection have been reported in association with waning of the immune response. While reinfection with viruses associated with systemic infections is uncommon, reinfection with viruses causing mucosal infections without viremia, such as respiratory syncytial virus, influenza, and coronavirus, is much more common [155]. In unvaccinated patients infected by coronaviruses, the mean time to reinfection ranged from 30 to 55 months, and 14% of patients infected had multiple reinfections with the same seasonal coronavirus strain [156]. In a more recent systematic review of the literature on more than 615,000 participants, reinfection was found to be an uncommon event and occurred in 0-1.1% [157]. The data from that review suggested that overall, naturally acquired SARS-CoV-2 immunity does not wane for about 10 months post-infection [157].

Complement Activation in COVID-19 and Gaucher Disease
The complement system comprises a group of liquid and cell membrane-associated proteins, which are mainly produced in the liver. However, certain brain cells, such as microglial cells, astrocytes, and neurons, are also involved in the direct synthesis of complement proteins [197][198][199]. Complement activation is a complex process, which largely occurs by the classical, alternative, and lectin pathways [200]. The classical pathway is activated by ligation of the IgG and/or IgM immune complexes (ICs) to their corresponding receptors and/or C1q on the cell surface. The alternative pathway is activated by binding of spontaneously activated C3 protein (C3b fragment) to host and non-host cell surfaces [201,202]. The lectin pathway is activated by the binding of the mannan-binding lectin (MBL) to mannose-containing carbohydrates or related ficolins to certain carbohydrates or acetylated structures [198,203,204]. Each of the complement activation pathways follows a series of reactions generating common key components known as C3 and C5 [205]. The downstream cleavage of C3 by the C3 convertases causes the formation of C3a and C3b [205]. Similarly, the downstream cleavage of C5 by the C5 convertases causes the formation of C5a and C5b [205]. C3a binds the C3aR receptor, and C5a binds C5aR1 and C5aR2 receptors [205]. C3b is a major opsonin that induces the tagging and phagocytic uptake of pathogens, and C5b initiates the terminal complement pathway, resulting in the formation of the membrane attack complex (MAC) composed of C5b, C6, C7, C8 and multiple C9 molecules [35,[205][206][207][208]. Several activated components of the complement system are essential for controlling cellular and metabolic functions in both visceral organs and the CNS [209][210][211][212][213]. However, the abnormal activation and production of complement components such as C3, C3b, iC3b, CR3, C5, C5a, and MAC/C5b-9 can contribute to visceral and CNS tissue damage in several diseases including, but not limited to, allergic diseases, cardiovascular diseases, age-related macular degeneration, systemic lupus erythematosus, traumatic brain injury, stroke, neuromyelitis optica spectrum disorders, and neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease and Huntington disease [214][215][216][217][218][219][220][221][222][223][224][225][226][227][228][229][230][231][232].
Complement activation has been observed in H5N1 and H1N1 influenza virus infections [233]. The sera and lung tissues of the MERS-CoV-infected, hDPP4-transgenic (hDPP4-Tg) mouse model showed complement activation and increased production of C5a and C5b-9, as well as increased viral replication [234,235]. Furthermore, targeting C5aR with anti-C5aR antibody treatment in the MERS-CoV-infected hDPP4-Tg mouse model decreased viral replication and damage to lung and spleen tissue [234,235]. The lung of the mouse-adapted SARS-CoV animal model has also demonstrated complement activation and higher virus replication [31]. To investigate whether SARS-CoV triggered complement activation in the mouse model, C3 -/and background matched control wildtype (WT) mice were infected with SARS-CoV-2 and their lung tissues analyzed for immune inflammation. The data showed that C3 -/mice infected with SARS-CoV-2 were protected against the development of respiratory dysfunction and lung damage when compared to WT mice [31].
SARS-CoV-2 infected primary human airway epithelial cells also caused complement activation and increased production of C3a [236]. C3aR and C5aR targeting in cellular models also showed protection from lung tissue damage [236]. Lung and skin tissues and sera from patients with severe COVID-19 have shown increased complement activation and massive generation of C5a, C5b-C9, and C5aR1 in blood and pulmonary myeloid cells [209]. Studies have shown that SARS-CoV-2 induced hyperinflammation is linked to the development of acute respiratory distress syndrome (ARDS), systemic clotting, and a variety of cutaneous manifestations in patients with COVID-19 [80][81][82][237][238][239][240][241]. Strikingly, administration of C3 (AMY101) or C5 (eculizumab) targeting drugs have shown marked reduction in the SARS-CoV-2-induced development of severe disease symptoms and lung damage in COVID-19 patients [80][81][82]. These data suggest that complement activation and the resultant activation of the C5a-C5aR1 axis is critical for propagating the disease complications in patients with COVID-19.

Complement Activation Is Linked to the Increased Synthesis of Glucosylceramide Synthase Enzymes and Excess Production of Sphingolipids in COVID-19 and GD
Several studies have shown that in vivo and ex vivo stimulation of immune cells with immune complexes (e.g., IgG and IgM ICs) and complement activation products (e.g., C3, C3a, C3b, and C5a) can cause excess secretion of lysosomal enzymes (Table 7). Sphingolipids are ubiquitous components of the plasma membrane of eukaryotic cells and are essential for controlling cell proliferation, survival, and death [4,307]. However, prolonged overabundance of sphingolipids is detrimental, as seen in several lysosomal storage disorders such as GD, Fabry disease, Tay-Sachs disease, Sandhoff disease, Krabbe disease, and Niemann-Pick disease [308]. GC is an important sphingolipid, representing the backbone of more than 450 structurally different glycosphingolipids. Activation of GCS, the enzyme that places a glucosyl moiety onto ceramide, is the first pathway-committed step in the production of more complex GSLs, such as lactosylceramide and gangliosides [309].

Complement Activation Products Cells Enzyme Secretion References
IgG and IgM-IC PMNs R Alkaline phosphatase, Acid phosphatase, and β -Glucuronidase [327] BSA-anti-BSA M
Sera and lung tissues of the MERS-CoV infected hDPP4-transgenic (hDPP4-Tg) mouse model showed increased concentrations of the C5a and C5b-9, as well as the overexpression of caspase-1 and IL1β. Furthermore, targeting C5aR1 by using anti-C5aR1 antibody treatment in the MERS-CoV-infected hDPP4-Tg mouse model caused decreased viral replication and reduced expression of IL1β and caspase-1, as well as the protection from cell death and tissue damage (e.g., lungs and spleen) [234,235]. A mouse-adapted SARS-CoV animal model showed complement activation is linked to the increased production of proinflammatory cytokines and chemokines, and the massive lung infiltration of inflammatory subsets of immune cells. C3 -/mice infected with SARS-CoV-2 had marked reduction in the production of IL1α, IL6, TNFα, CXCL2, GCSF, and decreased lung infiltration of inflammatory subsets of immune cells (e.g., MOs and PMNs), as well as the significant protection against the development of respiratory dysfunction and lung damage when compared to control WT mice.
SARS-CoV-2 infected primary human airway epithelial cells also caused increased production of C3a, IL1α, IL6, CCL2, and CCL5 [236]. C3aR and C5aR targeting in these mice and a cell model showed pronounced reduction in the development of immune inflammation and decreased lung tissue damage [236]. Furthermore, lung, skin, and sera specimens from patients with severe COVID-19 infection have shown massive generation of C5a, C5b-C9, and C5aR1 with myeloid cells infiltration and pro-inflammatory cytokines production (Table 1). Strikingly, administration of C3 (AMY101) and C5 targeting drugs (eculizumab) resulted in marked reduction of pro-inflammatory cytokines and improved lung function in SARS-CoV-2-induced COVID-19 [80][81][82]. Similarly, GC-induced activation of complement and the resultant production of C5a caused overproduction of chemokines and growth factors, leading to increased tissue recruitment of M  [64] s, DCs, and T cells eventually led to the production of inflammatory cytokines in mouse models and patients with type 1 GD [66,67,121,161]. Additionally, massive generation of chemokines (e.g., CCL2, CCL3, and CCL5), growth factors (e.g., MCSF and TGFβ), activation of the microglial cells and astrocytes, and the immense generation of pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL1α, IL1β, and IL6), reactive oxygen species (ROS), nitric oxide (NO), and their combined impact inducing neuronal loss and early death were observed in Gba flox/flox ; nestin-Cre mice, K14-lnl/lnl mice, 4L;C*, C57BL/6J-Gba tm1Nsb and the conduritol B epoxide-induced chemical model of nGD [167]. Type 2 and Type 3 human nGD display microglial cell activation and upregulation of pro-inflammatory cytokines (e.g., TNF α, IL1β, and IL6) that lead to the loss of neurons and to early death in patients with Type 2 and Type 3 nGD [167,172]. Furthermore, data from the Gba1 9V/mouse model, conduritol B epoxide-induced GCase-targeted mouse model and human cells, and iPSCs-derived macrophages from Type 1, Type 2 and Type 3 nGD patients, showed that the C5a-C5aR1 axis causes overgeneration of chemokines and growth factors. This leads to the increased recruitment and/or activation of innate and adaptive immune cells in visceral organs and microglial cells and/or neurons in the brain that causes overproduction of pro-inflammatory cytokines, leading to the development of severe and chronic visceral and brain tissue inflammation in GD [126]. Further, we and others have shown that genetic deficiency or pharmaceutical targeting of C5aR1 in GD mouse models and human cell model of GD resulted in marked reduction in the production of CCL chemokines (e.g., CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL9, CCL12, CCL17, CCL18, CCL22), CXCL chemokines (e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL19, CXCL11, and CXCL13), and growth factors (e.g., MCSF, GCSF, and GMCSF). In addition, a decreased tissue recruitment of immune cells (e.g., M s, DCs, CD4 + T cells), marked reduction in generation of the pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL1α, IL1β, IL6, IL12p40, IL12p70, IL17,IL18, IL21, and IL23) and protection of lung, liver, and spleen disruption was de,omnstrated [79,258]. These findings suggest that targeting the C5a-C5aR1 axis could control the SARS-CoV-2/GC-induced excess production of chemokines, tissue recruitment of immune cells, pro-inflammatory cytokines production and lessen the resultant tissue damage in COVID-19 and GD patients.

Discussion
SARS-CoV-2-induced increased production of complement activation products such as C5a, C5b-C9, and C5aR, which are linked to the increased tissue recruitment and activation of MOs, M  s, DCs, PMNs, CD4 + T cells, CD8 + T cells, and the extensive production of pro-inflammatory cytokines, chemokines, and growth factors (Tables 1 and 2). In addition, excess synthesis of GCS and the corresponding glycosphingolipids have been observed in COVID-19 [1][2][3][4]. Moreover, drugs targeting complement activation at the C5a-C5aR1 stage in the complement pathway and drugs targeting GCS have shown marked reduction in the replication of SARS-CoV-2, as well as the SARS-CoV-2-mediated induction of innate and adaptive immune inflammation that leads to the tissue destruction and death in patients with COVID-19 [78,[80][81][82]236].
Similarly, in vitro, ex vivo, and in vivo studies performed with the Gba1 mutant mouse models and for patients with GD have shown a link between the upregulation of C1q, C3, C4b, C5a, C5aR1 and the increased production of GC, excess recruitment and activation of innate and adaptive immune cells, and overproduction of pro-inflammatory cytokines, chemokines, and growth factors in many tissues in GD (Tables 3 and 4). Studying the mechanistic connections between the complement-sphingolipid axis in the induction of tissue inflammation in GD, we and others have found that massive GC storage causes the development of GC-specific IgG autoantibodies that bind to GC and produce the GC-specific IgG-ICs (GC-ICs) [258,348,349]. Such ICs drive complement activation, local production of C5a, and the activation of C5aR1 in Gba1 mutant (Gba1 9V/-) mouse model and patients with GD [79,126,258]. The activation of the indicated C5a-C5aR1 axis causes the excess cellular production of GCS and GC, innate and adaptive immune cell activation, and the development of tissue inflammation in GD [79,126,258]. Furthermore, we and others have shown that genetic deficiency or pharmaceutical targeting of C5aR1 in mouse models and patients with GD reduces generation of GCS-glycosphingolipid, inhibits the production of pro-inflammatory cytokines, chemokines, and growth factors, tissue recruitment of innate and adaptive immune cells, and protects the lung, liver, and spleen tissues from damage [79,258].
Several vaccines and alternative treatments have shown poor efficacy and or immunity against SARS-CoV-2 (Table 5). Strikingly, agents targeting C5-C5a-C5aR1 or glycosphingolipid-lowering therapies have been linked to the disruption of the SARS-CoV-2 replication and the suppression of the SARS-CoV-2-induced activation of immune inflammation in COVID-19 [24,78,350,351]. However, the mechanism by which targeting C5-C5a-C5aR1 or GCS-glycosphingolipid pathways inhibit the viral replication, and the induction of immune inflammation remains unknown; thus, additional in vitro, ex vivo, and in vivo studies on different variants of SARS-CoV-2 (e.g., Alpha, Beta Delta, BA.4, and BA.5)induced COVID-19 are required. Studies to test whether these SARS-CoV-2 variant-induced COVID-19 triggers the excess synthesis of GCS and the corresponding development of glycosphingolipids, glycosphingolipids-specific IgG antibodies, and complement activation in patients with COVID-19 and GD patients with COVID-19 are also needed. Furthermore, large scale experimental and clinical trials are critical to test whether combined targeting of complement at the level of C5a-C5aR1 and the GCS-glycosphingolipid pathway can affect the disease severity and/or death in patients with COVID-19 and in GD patients with COVID-19. Figure 1 provides a summary of the complement-glycosphingolipid axis, as supported by study findings from patients and mouse models for COVID-19 and GD reviewed herein. It is suggested that SARS-CoV-2 infection triggers the development of SARS-CoV-2 IgM-ICs and the SARS-CoV-2 and/or GSL; GC-specific IgG1-ICs. The binding of such SARS-CoV-2 IgM-ICs to FcµR and SARS-CoV-2 IgG1-ICs/GC-specific IgG1-ICs to activating FcγR, and the direct binding of SARS-CoV-2 to mannose-binding lectin (MBL-MASP 1-2) lead to the massive production of C5a in patients with COVID-19 and GD patients with COVID-19 ( Figure 1A,B). The interaction of such C5a to its C5aR1 receptor triggers GC synthase (GCS)-mediated excess synthesis of GSLs, which promotes the SARS-CoV-2 growth and replication ( Figure 1C) and over production of the cytokines, chemokines, and growth factors listed in Tables 1-4 and shown in Figure 1D. These pro-inflammatory mediators cause increased tissue infiltration and activation of several of the immune cells, i.e., MO, M , DC, PMN, and T cells, additional generation of pro-inflammatory cytokines ( Figure 1E) and lead to the tissue damage in patients with COVID-19 and GD with COVID-19 ( Figure 1F). Hence, targeting the C5-C5aR1-GCS glycosphingolipid pathway could protect the SARS-CoV-2-induced development of s severe form of the disease in patients with COVID-19 and GD patients with COVID-19. These findings also open up the exciting possibility of using drugs already available that effectively target the C5a-C5aR1 axis at the level of C5 (e.g., Eculizumab), which prevents C5 from being cleaved into C5a and C5b and is currently used for treating patients with Atypical hemolytic uremic syndrome and Paroxysmal Nocturnal Hemoglobinuria) [352,353]. Similarly, C5a-suppressing oral treatments (e.g., Avacopan, approved for the treatment for anti-PMN cytoplasmic antibody-associated vasculitis) and the pharmaceutical targeting of the GCS (e.g., Venglustat, which inhibits the synthesis of glycosphingolipids) can be used as potential approaches to control the tissue inflammation, organ failure, and death in patients with COVID-19 and GD patients with COVID-19 experiencing uncontrolled infections with different variants of SARS-CoV-2 (e.g., Alpha, Beta Delta, BA.4, and BA.5) [354,355]. From a clinical standpoint, this review emphasizes the possible role for medications targeting the complement-glycosphingolipid pathway as an additional anti-inflammatory therapeutic strategy to ameliorate disease progression in COVID-19 patients, patients at high-risk of developing COVID-19 or COVID-19 reinfection, and GD patients with COVID-19 disease. In sum, these immune strategies may be useful in patients for whom treatments and vaccines cannot be easily used, for example, patients at risk of developing severe, treatment-related adverse events, those with vaccine-related serious adverse events, or in patients with reinfection due to the immunity waning phenomenon.