S100A9: The unusual suspect connecting viral infection and inflammation

Abstract

The study of S100A9 in viral infections has seen increased interest since the COVID-19 pandemic. S100A8/A9 levels were found to be correlated with the severity of COVID-19 disease, cytokine storm and changes in myeloid cell subsets. These data led to the hypothesis that S100A8/A9 proteins might play an active role in COVID-19 pathogenesis. This review explores the structures and functions of S100A8/9 and the current knowledge on the involvement of S100A8/A9 and its constituents in viral infections. The potential roles of S100A9 in SARS-CoV-2 infections are also discussed.

Introduction

Thirty-five years ago, Odink and colleagues (1) purified a complex of two proteins of masses 8 and 14 kDa, respectively, from stimulated mononuclear cell cultures using an antibody to macrophage migration inhibition factor (MIF). These proteins were named MIF-related proteins (MRP-8 and MRP-14). They are now referred as S100A8 (MRP-8, calgranulin A; A8, CP-10) and S100A9 (MRP-14, calgranulin B; A9). The S100A8/S100A9 heterodimer is often called calprotectin because of its protective anti-microbial function. S100A8/A9 was later identified as the cystic fibrosis (CF) antigen, a protein elevated in the serum of CF patients (2). S100A8 and S100A9 form a subset of the S100 family of calcium-binding proteins. These intracellular proteins control protein phosphorylation, various enzymatic activities, Ca²⁺ homeostasis, activation of NADPH oxidase and intermediate filament polymerization (reviewed in (3)). S100A8 and S100A9 are primarily expressed by neutrophils and monocytes but are generally not expressed in tissue macrophages (4). However, S100A8 and S100A9 are inducible proteins expressed in exudate macrophages following infection with pathogens (5). Apart from myeloid cells, a recent study has delineated the roles of S100A8 and S100A9 produced from airway epithelial cells in regulating macrophage function during chronic obstructive pulmonary disease (COPD) (6). S100A8 and S100A9 exist as non-covalently bonded homodimers. In addition, S100A8 and S100A9 form a noncovalent heterodimer called S100A8/A9 or calprotectin (3). Ca²⁺ promotes heterotetramer formation of calprotectin and S100A9/A9 (7).

Structure

All S100 proteins have a conserved classical EF-hand and a second atypical domain with 14 rather than 12 residues with Ca²⁺-binding domains (8). The two EF-hand domains are separated by a hinge region that differs between members of the S100 protein family. The structurally divergent domains at the C-terminus and in a “hinge” region separating the EF-hands confer functional specificity to each S100 protein (9–11). Purified recombinant S100A8 and S100A9 spontaneously form homodimers regardless of the presence of divalent cations (12). When co-expressed, S100A8 and S100A9 spontaneously form homodimers and the heterodimer S100A8/A9 inside cells, the latter been the preferred form (10, 11). There are two isoforms of human S100A9, full-length and truncated (Δ5-S100A9), the latter generated by translation from an alternate start site at methionine 5. Truncated S100A9 accounts for ~30% of total S100A9 in neutrophils and does not form heterodimers with S100A8 (13). Structural studies of S100 proteins indicate at least three recognition sites within two distinct surfaces that accommodate multiple binding partners, including the hinge domain between the Ca²⁺-binding regions (14). This explains why some functions of S100A8 and S100A12 reside within this divergent domain (15). Furthermore, S100A9 has an extended C-terminal domain consisting mainly of hydrophilic amino acid residues highly homologous to the neutrophil immobilizing factor (NIF) peptides (16). This peptide reduces migration and phagocytosis of adherent macrophages (17) and pain responses in inflammation (17, 18). Therefore, the C-terminal domain of S100A9 is a likely candidate for binding to its receptors.

S100A8 and S100A9 are presumed to bind to RAGE (19), the scavenger receptor (CD36) (20), CD33 (21), CD85i (22), or Toll-like receptor 4 (TLR4, K_D=3.2 nM) (23). The specificity of the binding to TLR4 was confirmed using anti-TLR4 antibodies, while the loss of binding following heat denaturation and the absence of the effect of polymyxin B confirmed that the binding was not due to LPS contamination (23, 24). Loes et al. demonstrated by complementation experiments that TLR4 and S100A9 coevolved, suggesting that TLR4 is the principal receptor for S100A9 (25). However, the domains of S100A9 interacting with its receptors remain unknown.

Activity.

By the mid-’90s, S100A8/A9 had been detected in various inflammatory settings, but it was unclear if it was merely a marker of inflammation or if it played an active role in immunity. Since then, studies have revealed that S100A8 and S100A9 are amongst the most potent chemotactic factors for neutrophils and monocytes, with chemotactic activities in the 10⁻¹⁰ M (26–28). Circulating neutrophils, macrophages, and monocytes massively express and secrete S100A8, S100A9 and S100A8/A9 to modulate inflammatory processes (5, 11, 29–31). On one hand, anti-inflammatory factors like glucocorticoids and IL-10 induce S100A8 expression (32, 33). Also, S100A8 induces the expression of IL-10 (34) and inhibits mast cell degranulation and FcεR-crosslinking-induced cytokine secretion (35, 36). In addition, mice deficient in S100A8 have aggravated collagen-induced arthritis and imiquimod-induced psoriasis, suggesting an anti-inflammatory function for this protein (37, 38). On the other hand, the secretion of S100A9 and S100A8/A9 is promoted by adhesion to endothelium and pro-inflammatory factors like monosodium urate crystals, TNF and IL-1, suggesting a pro-inflammatory function (11, 29, 30).

S100A9 is preferentially secreted during inflammation; there are approximately four times more S100A8/A9 and 60 times less S100A8 secreted in response to LPS than S100A9 (39). S100A9 induces neutrophil adhesion to extracellular matrix proteins and prolongs CD11b activation, leading to enhanced transendothelial migration (40, 41). At the inflammatory site, S100A9 induces the production of nitric oxide and reactive oxygen species and the degranulation of neutrophils in a process involving JNK and p38 (5, 42–45). S100A9 is also one of the most potent stimulators of neutrophil phagocytosis and bacterial killing following phosphorylation of syk, Erk1/2 and Akt (43), and it activates the translocation of NF-ĸB to the nucleus, leading to the secretion of TNF, IL-1β and IL-6 (45). S100A9 is also secreted by activated platelets and is involved in thrombosis (46). S100A8 and S100A9 promote inflammation in models of gout, arthritis, psoriasis, bacterial and viral lung inflammation, and malaria and leishmania infections (5, 29, 37, 41, 47, 48). Other studies have shown a role for these proteins in pain, myocardial infarction, traumatic brain injury, stroke, asthma, glomerulonephritis, and Alzheimer’s disease, to name a few (49–54). Thus, S100A8 and S100A9 are important regulators of inflammatory response that shape inflammation status during various disease states. Interestingly, apart from its role as an immune regulator, calprotectin may also have a direct role as an anti-microbial factor since it inhibits the growth of Candida spp, Cryptococcus neoformans, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella spp and Alcaligenes spp (55–58).

It now appears that S100A8 and S100A9 have opposite activities. S100A9 promotes inflammation by enhancing phagocyte migration and inducing the secretion of pro-inflammatory cytokines, tissue-degrading enzymes and ROS. S100A8 presumably counterbalances these functions with its anti-inflammatory activities. Secretion of S100A9 increases over time during an inflammatory reaction, reaching maximal concentrations 24h to 48h after onset at a time when TNF has subsided (5, 29). Consequently, S100A9 probably enhances the inflammatory response.

S100A9 and viral infection

Human immunodeficiency virus-1 (HIV-1):

HIV-1 is mainly transmitted by vaginal or anal sexual contact across mucosal surfaces and usually first infects resident myeloid cells. From the earliest stages of HIV-1 infection, the inflammatory cascade drives dendritic cells to secrete calprotectin (Figure 1). In addition, calprotectin secretion is accompanied by increased production of extracellular vesicles (eVs) by CD4 T lymphocytes in contact with HIV-1 (C. Gilbert, unpublished results) as well as enrichment of miR-155 in eVs (59). In 1995, calprotectin was described as a non-specific agent involved in epithelial cell defense mechanisms against HIV-1 (60). In the same year, it was denoted as a marker of HIV-1 disease stage (61). However, very few studies have focused on the roles of calprotectin and S100A9 in the inflammation associated with HIV-1 disease. Serological analysis has shown increased calprotectin levels in HIV-1-positive individuals exhibiting advanced immune deficiency before HAART introduction (61). Increased S100A9 and calprotectin levels have also been detected in HIV-1-positive patients with opportunistic infections (61). In the mid-1990s, higher calprotectin levels were observed in the serum of HIV-1-positive and AIDS patients (62). These higher levels correlated with comorbidity and opportunistic infections associated with HIV-1 infection (61). In a study of HIV-1 infected, treatment-naïve children under 12, fecal calprotectin levels were higher in patients with lower CD4 T-cell counts (63). These results were confirmed in a recent study (64). The increase in fecal calprotectin is due to the pro-inflammatory state of the intestinal mucosa (65). In addition, limited immune restoration after ART initiation was linked to higher fecal calprotectin levels. Conversely, lower fecal calprotectin was associated with a better immune system restoration (66). Fecal calprotectin is also associated with the presence of plasma inflammation markers like IL-6, high-sensitivity C-reactive protein, soluble tumor necrosis factor receptor-II (sTNFR-II) and soluble vascular cell adhesion molecule-1 (sVCAM-1) (64). High levels of S100A9 protein in the breast milk of women with HIV-1 who transmit the virus to their infants through breastfeeding suggests a role for this protein in the transmission of HIV-1 through breastfeeding (67). In vitro studies subsequently showed that HIV-1 transcription and viral replication may be enhanced by S100A8, S100A9, and calprotectin through activation of the transcription factor NF-κB (68). The S100A8 protein produced by neutrophils and macrophages is found in cervicovaginal secretions and activates HIV-1 replication (69). Interestingly, neutrophils release calprotectin (and presumably S100A9) in response to the TLR7/8 agonist R848, suggesting that single-stranded RNA promotes its secretion (C. Gilbert and P.A. Tessier, unpublished observations). Also, the double-stranded RNA produced during infection leads to increased production of S100A8 by macrophages (70). These observations were corroborated in latently infected macrophages, where S100A8 reactivated virus production (71). However, a study has shown enhanced LPS-mediated response of neutrophils derived from HIV-1 positive individuals compared to HIV-1 negative individuals [82]. The same study also showed that in contrast to LPS response, neutrophils from HIV-1 positive individuals had diminished calprotectin-mediated response in inhibiting the oxidative metabolism of neutrophils [82]. Despite these observations, the significance and roles of calprotectin and S100A9 in plasma or intestinal tissues of HIV-1-positive individuals remain uncertain.

Figure 1:

HIV-1 infects resident myeloid cells in the mucosa, leading to the secretion of calprotectin and S100A9 by dendritic cells. High levels of calprotectin are also detected in the blood. S100A9 and calprotectin activate HIV-1 transcription and replication in infected CD4 T lymphocytes.

Calprotectin and S100A9 may also have a beneficial effect on the immune response in the context of HIV-1 infection. For example, calprotectin in oral mucosa inhibits the development of Candida albicans co-infection (72). Furthermore, S100A9 negatively regulated HIV-1 reverse transcription in Langerhans cells and prevented infection (73). In addition, binding of S100A9 to NK cells enhances their anti-HIV-1 activity (74). This translates into an improved cytotoxic effect against infected CD4 T lymphocytes (22) and increased IFN-γ production (74). These findings were corroborated in a study which showed that S100A8, S100A9 and calprotectin activate HIV-1 transcription by promoting the binding of the transcription factor NF-κB to the LTR region of the HIV-1 genome (68). In addition, infection of Jurkat cells in the presence of calprotectin increases virion production (68). In summary, calprotectin is overexpressed in the serum and genital epithelium of individuals with HIV-1, and it modulates HIV-1 infection by directly regulating its transcriptional activity or augmenting anti-HIV-1 immuno-activity.

Lung infection

S100A9 regulates three fundamental mechanisms contributing to lung inflammation in infection. First, it increases the number of myeloid cells in circulation. S100A9 has been shown to induce the differentiation of acute myeloid cells and their growth arrest in human and mouse models of acute myeloid leukemia (75). In myeloid cells, S100A9 activates the TLR4/MyD88 pathway, leading to the phosphorylation of p38, ERK1/2, and JNK, which in turn activate CREB, c-JUN, and NF-κB (75). Moreover, the addition of S100A9 stimulates the differentiation of neutrophils, monocytes and macrophages in short-term cell cultures of bone marrow cells (P.A. Tessier, unpublished observations). In consequence, elevated levels of S100A9 are likely to lead to increased myelopoiesis. Also, when injected intravenously, S100A9 induces leukocyte egress from the bone marrow (29). As egress occurs within thirty minutes, it is likely a direct effect not requiring de novo protein expression. Thus, elevated S100A9 in the serum leads to increased myelopoiesis and neutrophilia in the blood. Consistent with this, S100A9 maintains circulating neutrophil numbers and recruitment to streptococcal pneumonia-infected murine lungs, principally by regulating G-CSF production (76).

A second mechanism of action of S100A9 is the promotion of leukocyte migration to the lung in vivo (5, 26, 39). Leukocyte migration in the lung differs from the classical tethering-rolling-firm adhesion mechanism. It does not require selectins, as rolling does not occur in this tissue. The principal site of leukocyte migration in the lung is the capillary bed, as the diameter of neutrophils (13.7 μm) is bigger than that of capillaries (ranging from 2 to 14 μm) (77). Consequently, neutrophils do not roll and rely primarily on integrins for their migration in the lung (78). The CD18 integrin Mac-1 (CD11b/CD18) is particularly important for neutrophil recruitment in the lung as blocking Mac-1, but not lymphocyte function-associated antigen-1, inhibits neutrophil migration (79, 80). S100A8 and S100A9 stimulate neutrophil adhesion to endothelial cells and fibrinogen in a Mac-1 integrin-dependent manner (26, 81). S100A9 also promotes neutrophil adhesion to fibronectin, a major protein of the interstitial space (40). In addition, the presence of S100A9 in the blood is sufficient to stimulate transendothelial migration, even in the absence of chemotactic factors to attract and promote the extravasation of neutrophils (40, 41). Interestingly, S100A9 does not stimulate the expression of adhesion molecules on endothelial cells or increase endothelial cell permeability (41). However, S100A9 prolongs the adhesion of neutrophils and favours their migration through prolonged stimulation of the β2 integrin CD11b (40, 41). Thus, the presence of S100A9 in the blood likely leads to increased migration of myeloid cells to the lung. S100A9 provokes cytokine and chemokine secretion from preformed stores, thereby contributing to the leukocyte infiltration (82). Moreover, S100A9 induces CXCL10 expression, contributing to neutrophilia in the airways (83).

The third mechanism of action of S100A9 is the induction of pro-inflammatory cytokine production (5, 45, 84). S100A9 deficiency is associated with increased cytokine levels and aggravated lung pathology in mice infected with S. aureus. However, this might be due to increased bacterial growth due to the lack of calprotectin (85). The pro-inflammatory activity of S100A9 during respiratory virus infection has been well studied, as described below.

Respiratory virus infection

Respiratory viruses like influenza A virus (IAV), human respiratory syncytial viral (RSV), and SARS-CoV-2 are clinically important viruses that cause mortality and morbidity in infected populations. These viruses trigger hyper-inflammatory lung diseases like pneumonia and bronchiolitis. Studies with these viruses have revealed that S100A9 is a damage-associated molecular pattern (DAMP) released from infected cells to confer its functional activity. Both IAV [31] and RSV (86) infection trigger the release of S100A9 from infected cells. S100A9 has also been detected in patients infected with IAV and RSV (87, 88). During IAV and RSV infection, S100A9 acts as a pro-inflammatory DAMP that amplifies the inflammatory response that culminates in exaggerated airway inflammation and exacerbates lung diseases (5, 86).

Besides RSV and IAV, recombinant SARS-CoV-2 spike and nucleocapsid proteins increase S100A9 expression in human primary PBMCs (89). COVID-19 is the disease caused by SARS-CoV-2. Plasma levels of calprotectin are elevated in patients with COVID-19 and correlate with disease severity and thrombotic complications (90). Moreover, deposition of S100A8/A9 on lung vessel walls was observed in lungs from fatal COVID-19 cases (90). S100A9 was also found to be upregulated in PBMCs from COVID-19 patients (89), and S100A9 is the most increased inflammatory mediator in severe COVID-19 patients compared to healthy controls (89). Thus, respiratory virus infection triggers calprotectin and S100A9 release to promote inflammation. How calprotectin and S100A9 are released during infection is still unknown. It could be envisioned that lytic cell death comprising of pyroptosis of myeloid cells like macrophages can facilitate the release of calprotectin and S100A9 from infected cells. IAV (91), RSV (92), and SARS-CoV-2 (93) trigger pyroptosis by forming plasma membrane pores comprising MLKL and Gasdermin-D. These pores are involved in ion transport and the release of cytokines like IL-1β and IL-18. It is plausible that these pores formed during virus infection are utilized by calprotectin and S100A9 for extracellular release. This hypothesis is supported by the facts that pyroptosis inducing agonists like H₂O₂ induce S100A8/A9 release from neutrophils (11), that auto-inflammatory patients have increased serum IL-1β and S100A8/A9 (94), and that mutations to the pyrin-binding protein proline-serine-threonine phosphatase interacting protein 1 lead to increased inflammasome activation and secretion of IL-1β and S100A8/A9 (11, 95, 96).

S100A9 contributes to respiratory virus pathogenesis by inducing pro-inflammatory cytokine production (5, 45, 84, 97). During IAV infection, S100A9 expression is induced via the DDX21-TRIF pathway, and the secreted S100A9 activates the TLR4-MyD88 pathway (5). Similarly, TRIF pathway is utilized for S100A9 induction during RSV infection (86). S100A9 plays a critical role in exaggerated inflammation since blocking the pro-inflammatory activity of S100A9 drastically reduces airway inflammation and IAV- and RSV-associated immuno-pathology (5, 86). The pro-inflammatory activity of S100A9 is evident from the expression/production of pro-inflammatory cytokines like TNF and IL-6 in macrophages treated with purified S100A9 protein (5). Similarly, intratracheal administration of purified S100A9 protein to mice triggers the expression/production of TNF and IL-6 in the lung (5).

As described above, SARS-CoV-2 infection promotes the production of S100A9. Interestingly, S100A8 and S100A9 contribute to SARS-CoV-2 pathogenesis by aberrantly activating the “detrimental” neutrophils that cause severe tissue injury (98). In addition, recombinant S100A9 enhances IL1B mRNA expression induced by SARS-CoV-2 S protein in PBMCs (89).

One of the hallmarks of COVID-19 pathogenesis is a patient succumbing to thrombosis. S100A8/A9 heterodimer in the serum of COVID-19 patients is related to an increased risk of dysregulated thrombotic events (99). Hyperactive coagulation is associated with most severe COVID-19 cases (100). Additionally, D-dimer level is associated with disease severity and is a prognostic biomarker for in-hospital death in subjects admitted due to COVID-19 (101). Circulating activated platelets and platelet-leukocyte aggregates are increased in infectious conditions such as sepsis, influenza, and COVID-19, with the increase in these levels correlating with disease severity (102–104). Moreover, procoagulant platelets and microvesicles are observed in COVID-19, but the mechanisms triggering their formation are unknown (105).

Recent work has demonstrated that S100A9 may contribute to inflammation-associated thrombosis (90). Interestingly, platelets have mRNA for S100A9 and express and secrete S100A9 protein after vascular injury (46). S100A9 promotes thrombus formation via binding to CD36 receptor (but not TLR4) on platelets (46). Moreover, blocking S100A9-CD36 interaction suppresses thrombus formation (46, 52). In addition, S100A8/A9 was demonstrated recently to accelerate fibrin generation and induce alpha granule secretion (90). However, S100A8/A9 does not induce fibrinogen binding and platelet aggregation (90), suggesting once again that S100A9 homodimers are more biologically active than the heterodimer S100A8/A9. However, some facts contradict such a role for S100A9. Indeed, there is a homology between the C-terminal ‘tail’ region of S100A9 and high molecular weight kininogen, which binds to negatively charged surfaces such as kaolin (106). S100A9 also binds to kaolin, and this binding is competitively inhibited by high molecular weight kininogen. Furthermore, S100A9 and the tail peptide inhibit the coagulation cascade (106). Thus, S100A9 functions in platelet activation and thrombus formation in lung viral infections remain to be clarified.

Concluding remarks

We have attempted to highlight the role of S100A9 and S100A8 during HIV-1 and three respiratory viruses (IAV, RSV, and SARS-CoV-2). However, apart from these viruses, additional viruses like rhinoviruses, bovine viral diarrhea virus, herpesvirus, and Coxsackievirus also induce S100A8/A9 and their expression regulates infectivity. We chose HIV-1, IAV, RSV, and SARS-CoV-2 for this review since they are timely, and detailed mechanistic studies have been performed to delineate the role of S100A9/A8 during infection. It is becoming clear that S100A9 is secreted by neutrophils and monocytes during viral infections. Extracellular S100A9 promotes neutrophil and monocyte migration and activation, leading to cytokine release and netosis. These activities contribute to the pathogenic inflammation associated with viral infection.

Literature related to S100A8/A9 can be confusing, with reported contradictory activities often due to inadequate reagents and methods. For example, as calprotectin is a non-covalently bound heterodimer, purified calprotectin preparations are always contaminated with S100A8 and S100A9 homodimers. Thus, it impedes interpreting results with purified proteins since reported activity with purified proteins could be due to S100A8/A9 heterodimers or S100A8 / S100A9 homodimers. Pure recombinant protein preparations, free of endotoxins, are also challenging to obtain, as S100A8 and S100A9 are lipophilic and bind LPS. Therefore, it is unsurprising that receptors binding lipids like TLR4 and CD36 have been suggested as receptors for S100 proteins. RAGE (another lipophilic receptor) is also supposed to bind to S100A9 and S100A8 (107), but other laboratories could not confirm these findings ((108) and P.A. Tessier, unpublished observations). Several labs also use quinoline-3-carboxamides as specific inhibitors of S100A9. However, quinoline-3-carboxamides block experimental autoimmune encephalomyelitis in S100A9 KO mice (109), and neutrophil adhesion and production of reactive oxygen species in the absence of S100A9 (110), demonstrating that this is not a specific inhibitor of S100A9. The functions of S100A8 and S100A9 were also examined in S100A9 KO and S100A8 mice. These mice were reported as quasi-S100A8/A9 KO because of their lack of expression of S100A8 proteins. However, S100A9 KO mice express low but detectable levels of S100A8 proteins and expression of S100A8 increases during inflammation (38). These shortcomings regarding the available reagents for the investigation of S100A8, S100A9 and calprotectin validate the need for further studies using various methods to confirm and expand our knowledge of their functions in inflammation.