A Potential Role of the CD47/SIRPalpha Axis in COVID-19 Pathogenesis

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Most SARS-CoV-2 infections are mild or even asymptomatic. However, a small fraction of infected individuals develops severe, life-threatening disease, which is caused by an uncontrolled immune response resulting in hyperinflammation. However, the factors predisposing individuals to severe disease remain poorly understood. Here, we show that levels of CD47, which is known to mediate immune escape in cancer and virus-infected cells, are elevated in SARS-CoV-2-infected Caco-2 cells, Calu-3 cells, and air−liquid interface cultures of primary human bronchial epithelial cells. Moreover, SARS-CoV-2 infection increases SIRPalpha levels, the binding partner of CD47, on primary human monocytes. Systematic literature searches further indicated that known risk factors such as older age and diabetes are associated with increased CD47 levels. High CD47 levels contribute to vascular disease, vasoconstriction, and hypertension, conditions that may predispose SARS-CoV-2-infected individuals to COVID-19-related complications such as pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury. Hence, age-related and virus-induced CD47 expression is a candidate mechanism potentially contributing to severe COVID-19, as well as a therapeutic target, which may be addressed by antibodies and small molecules. Further research will be needed to investigate the potential involvement of CD47 and SIRPalpha in COVID-19 pathology. Our data should encourage other research groups to consider the potential relevance of the CD47/ SIRPalpha axis in their COVID-19 research.

The first COVID-19 vaccines have been developed [2], and their roll-out has started in many countries. However, it will take a significant time until large parts of the global population will be vaccinated, and there is growing concern about the emergence of escape variants that can bypass the immunity conferred by the current vaccines and previous SARS-CoV-2 infections [5][6][7][8][9][10]. Thus, for the foreseeable future, there will be a need for improved COVID-19 therapies.
In a controlled open-label trial, dexamethasone reduced mortality in patients receiving oxygen with (from 41.1% to 29.3%) or without (from 26.2% to 23.3%) mechanical ventilation, but increased mortality in patients not requiring oxygen support [13]. Other immunomodulatory therapy candidates are being tested, but conclusive results are pending [11]. Further COVID-19 therapeutics under investigations include anticoagulants that target COVID-19-induced systemic coagulation and thrombosis (coagulopathy) [15].
However, it would be much better to have effective antiviral treatments that reliably prevent COVID-19 disease progression to a stage where immunomodulators and anticoagulants are needed. The antiviral drug remdesivir was initially described to reduce the recovery time from 15 to 10 days, and the 29-day mortality from 15.2% to 11.4% [11,16]. However, other trials did not confirm this and conclusive evidence on the efficacy of remdesivir remains to be established [11]. The JAK inhibitor baricitinib, which interferes with cytokine signaling, was reported to improve therapy outcomes in combination with remdesivir in a double-blind, randomized, placebo-controlled trial, in which patients were either treated with remdesivir plus baricitinib or remdesivir plus placebo [17]. Moreover, convalescent sera and monoclonal antibodies are under clinical investigation for COVID-19 treatment [18][19][20].
Ideally, antiviral therapies are used early in the disease course to prevent disease progression to the later immunopathology-driven stages [21]. However, only a small proportion of patients develop severe disease [21]. Therefore, a better understanding of the underlying processes is required to identify the patients who could develop severe disease as early as possible.
CD47 is the receptor of thrombospondin-1 (THBS1) and the counter-receptor for signal regulatory protein-α (SIRPα). CD47 interaction with SIRPα inhibits the activation of macrophages and dendritic cells, and thrombospondin-1/ CD47 signaling inhibits T cell activation [22,23]. Cellular surface levels of CD47 modulate immune responses in infectious diseases caused by parasites, bacteria, and viruses [22]. Typically, high CD47 levels prevent the immune recognition of virus-infected cells [22,24]. Moreover, cancer cells have been described to avoid immune recognition by upregulating CD47 [22]. Here, we investigated the potential role of the ubiquitously expressed cell surface glycoprotein CD47 in severe COVID-19.

Cell Culture
Calu-3 cells (ATCC, #HTB-55) were grown at 37 • C in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL of streptomycin. All of the culture reagents were purchased from Sigma. The cells were regularly authenticated by short tandem repeat (STR) analysis and were tested for mycoplasma contamination.
Primary human bronchial epithelial cells were purchased from ScienceCell. For differentiation to air−liquid interface (ALI) cultures, the cells were thawed and passaged once in PneumaCult-Ex Medium (StemCell Technologies, Vancouver, BC, Kanada) and then seeded on transwell inserts (12-well plate, Sarstedt, Nümbrecht, Germany) at 4 × 10 4 cells/insert. Once the cell layers reached confluency, the medium on the apical side of the transwell was removed, and the medium in the basal chamber was replaced with PneumaCult ALI Maintenance Medium (StemCell Technologies, Vancouver, BC, Kanada) including an antibiotic/antimycotic solution (Sigma Aldrich, Saint Louis, MO, USA) and MycoZap Plus PR (Lonza, Basel, Switzerland). Over a period of four weeks, the medium was changed and cell layers were washed with PBS every other day. Criteria for successful differentiation were the development of ciliated cells and ciliary movement, an increase in transepithelial electric resistance indicative of the formation of tight junctions, and mucus production.
Monocytes were infected at an MOI of 1 with SARS-CoV-2/FFM7 for 2 h. After infection, the cells were washed three times with PBS and subsequently cultivated in RPMI1640 (Gibco) supplemented with 100 IU/mL penicillin and 100 µg/mL streptomycin.

qPCR
SARS-CoV-2 RNA from cell culture supernatant samples was isolated using the AVL buffer and QIAamp Viral RNA Kit (Qiagen, Vienna, Austria) according to the manufacturer's instructions. SARS-CoV-2 RNA from the cell lysates was isolated using an RTL Buffer and the RNeasy Mini Kit (Qiagen, Vienna, Austria), according to the manufacturer's instructions. Absorbance-based quantification of the RNA yield was performed using the Genesys 10S UV−ViIS Spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA was subjected to OneStep qRT-PCR analysis using the Luna Universal One-Step RT-qPCR Kit (New England Biolabs, Hitchin, UK) and a CFX96 Real-Time System, C1000 Touch Thermal Cycler. Primers were adapted from the WHO protocol29 targeting the open reading frame for RNA-dependent RNA polymerase (RdRp): RdRP_SARSr-F2 (GTG ARA TGG TCA TGT GTG GCG G) and RdRP_SARSr-R1 (CAR ATG TTA AAS ACA CTA TTA GCA TA) using 0.4 µM per reaction. Standard curves were created using plasmid DNA (pEX-A128-RdRP) harboring the corresponding amplicon regions for the RdRP target sequence according to GenBank accession number NC_045512. For each condition three biological replicates were used. The mean and standard deviation were calculated for each group.

Data Acquisition and Analysis
Normalized protein abundance data from SARS-CoV-2-infected Caco-2 cells were derived from a recent publication [29] and are available from the PRIDE repository [30] (dataset identifier PXD017710). Data were subsequently normalized using summed intensity normalization for sample loading, followed by internal reference scaling and trimmed mean of M normalization. Mean protein abundance was plotted using the function ggdotplot of the R package ggpubr. p-values were determined by two-sided Student's t-test.
Raw read counts from SARS-CoV-2-infected Calu-3 cells were derived from a recent publication [31] via the Gene Expression Omnibus (GEO) database (accession: GSE147507) and were processed using DESeq2. Normalized gene counts were plotted using the function ggdotplot of the R package ggpubr. p-values were determined by two-sided Student's t-test.

Literature Review
Relevant articles were identified by using the search terms "CD47 aging", "CD47 hypertension", "CD47 diabetes", and "CD47 obesity" in PubMed (https://pubmed.ncbi. nlm.nih.gov accessed on 17 February 2021) on the basis of the principles outlined in the PRISMA guidelines (http://prisma-statement.org accessed on 17 February 2021). Articles in English were included in the analysis when they contained original data on the influence of aging, diabetes, diabetes, or obesity on CD47 expression levels and/or the relevance of CD47 with regard to pathological conditions observed in severe COVID-19. Two reviewers independently analyzed the articles for relevant information and then agreed a list of relevant articles.

Increased SIRPα Levels in SARS-CoV-2-Infected Monocytes
CD47 inhibits the activity of innate immune cells via the interaction with SIRPα [22,23]. Hence, we next investigated whether the SARS-CoV-2 infection of monocytes may impact SIRPα levels. SARS-CoV-2 did not result in a productive infection of primary human monocytes as indicated by a lack of an increase in genomic RNA levels ( Figure 2A). However, SARS-CoV-2 infection resulted in increased SIRPα levels in primary monocytes ( Figures 2B, S4 and S5). Hence, SARS-CoV-2 may interfere with both players of the CD47/SIRPα axis.  Figure S1. p-values were determined by two-sided Student's t-test. * p < 0.05, ** p < 0.01.  Figure S4. Quantification of the protein levels is provided in Figure S5.

CD47 and COVID-19 Risk Factors
To further investigate the potential role of CD47 in severe COVID-19, we performed systematic literature searches on the relationship of CD47 and the known COVID-19 risk factors of "ageing", "diabetes", and "obesity".

CD47 and Aging
The risk of severe COVID-19 disease and COVID-19 death increases with age [1]. A literature search in PubMed (https://pubmed.ncbi.nlm.nih.gov, accessed on 17 February 2020) using the terms "CD47" and "aging" resulted in 62 hits (Table S1). Eight of these articles contained information that supports a link between age-related increased CD47 levels and an elevated risk of severe COVID-19 ( Figure 3 and Table S1). One article suggested that alpha-tocopherol reduced age-associated streptococcus pneumoniae lung infection in mice through CD47 downregulation [33], which is in accordance with the known immunosuppressive functions of CD47 [22,23]. The remaining seven articles reported on age-related increased CD47 levels in vascular cells that are associated with reduced vasodilatation and blood flow (Table S1), as CD47 signaling inhibits NO-mediated activation of soluble guanylate cyclase and in turn vasodilatation [34,35]. As reduced vasodilatation can cause hypertension [36], we performed a follow-up literature search using the search terms "CD47 hypertension" (Table S2).
This resulted in 20 hits, including a further six relevant studies ( Figure 3B and Table S2). The evidence supporting a link between aging and/or hypertension and increased CD47 levels is summarized in Table 1. Table 1. Evidence supporting a link between aging and/or hypertension and increased CD47 levels.

Reference
Link between Aging and/or Hypertension and Increased CD47 Levels [33] CD47 downregulation may be involved in the alpha-tocopherol-mediated inhibition of age-associated streptococcus pneumoniae lung infection in mice [37] Blocking thrombospondin-1/CD47 signaling alleviates deleterious effects of aging on tissue responses to ischemia [38] CD47 null mice indicate that CD47 functions as a vasopressor [39] CD47-null mice are leaner-loss of signaling from the TSP1-CD47 system promotes the accumulation of normally functioning mitochondria in a tissue-specific and age-dependent fashion, leading to enhanced physical performance, lower reactive oxygen species production, and more efficient metabolism [40] High CD47 levels promote pulmonary arterial hypertension in the lungs from humans and mice [41] TSP1-CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction [42] Increased THBS1/CD47 signaling contributes to reduced skin blood flow and wound healing in aged mice [43] CD47 blocks NO-mediated vasodilatation [44] THBS1/CD47 signaling drives endothelial cell senescence [45] TSP1 promotes ageing-associated human and mouse endothelial cell senescence through CD47 [46] Increased CD47 expression causes age-associated deterioration in angiogenesis, blood flow, and glucose homeostasis [47] Increased CD47 levels in the lung of a sickle cell disease patient with pulmonary arterial hypertension relative to control tissues [48] Pulmonary hypertension reduced in a CD47-null mouse model of sickle cell disease [49] Anti-CD47 antibodies reversed fibrosis in various organs in mouse models Initial experiments showed that loss or inhibition of CD47 prevented age-and diet-induced vasculopathy and reduced damage caused by ischemic injury in mice [37]. CD47-deficient mice indicated that CD47 functions as a vasopressor, and the mice were also shown to be leaner and to display an enhanced physical performance and a more efficient metabolism [38,39]. In agreement, CD47 was upregulated in clinical pulmonary hypertension and contributed to pulmonary arterial vasculopathy and dysfunction in mouse models [40,41]. Age-related increased CD47 levels further affected peripheral blood flow and wound healing in mice [42] and NO-mediated vasodilatation of coronary arterioles of rats [43]. Moreover, thrombospondin-1/CD47 signaling was shown to induce ageing-associated senescence in endothelial cells [44,45] and age-associated deterioration in angiogenesis, blood flow, and glucose homeostasis [46].
Increased CD47 levels were also detected in the lungs of a sickle cell disease patient with pulmonary arterial hypertension, and vasculopathy and pulmonary hypertension were reduced in a CD47-null mouse model of sickle cell disease [47,48]. Finally, anti-CD47 antibodies reversed fibrosis in various organs in mouse models [49], which may be relevant in the context of COVID-19-associated pulmonary fibrosis [50].

CD47 and Diabetes
Diabetes has been associated with an increased risk of severe COVID-19 and COVID-19-related death [4]. A PubMed search for "CD47 diabetes" produced 47 hits, nine of which reported increased CD47 levels in response to hyperglycemia and/or diabetes ( Figure 4A and Table S3). Hyperglycemia protected CD47 from cleavage, resulting in increased CD47 levels [58][59][60][61]. In agreement, increased CD47 levels were detected in various cell types and tissues in rat diabetes models and diabetes patients [62][63][64][65][66] (Table 2). Therefore, diabetes-induced increased CD47 levels may interfere with the recognition of SARS-CoV-2-infected cells by the immune system [22,23] (Figure 4B). Table 2. Evidence supporting a link between diabetes and increased CD47 levels.

Reference
Link between Aging and/ or Hypertension and Increased CD47 Levels [58] Hyperglycemia protects CD47 from cleavage [59] Hyperglycemia protects CD47 from cleavage [60] Hyperglycemia protects CD47 from cleavage [61] Hyperglycemia protects CD47 from cleavage [62] CD47 is involved in pathophysiological changes in retinal cells in response to hyperglycemia in cell cultures and rats [63] Elevated CD47 mRNA levels both in the hippocampus and prefrontal cortex of a type-2 diabetes rat model [64] Increased levels of CD47 in epiretinal membranes with active neovascularization in proliferative diabetic retinopathy [65] Increased THBS1/CD47 signaling in bone marrow-derived angiogenic cells in a rat diabetes model [66] Increased diabetes-associated CD47 levels inhibit angiogenesis and wound healing in a diabetes model in rats

CD47 and Obesity
As obesity is another risk factor for severe COVID-19 [4], we also performed a PubMed search for "CD47 obesity", which resulted in eight hits, two of which provided potentially relevant information (Table S4). The results indicated that CD47-deficient mice were leaner, probably as a consequence of elevated lipolysis [67,68]. Hence, low CD47 levels may be associated both with lower weight and increased immune recognition of virus-infected cells [22,23,67,68], but there is no direct evidence suggesting that obesity may also directly increase CD47 expression. However, obesity may at least indirectly contribute to enhanced CD47 levels as a risk factor for diabetes and hypertension [4].

Discussion
Here, we show that the levels of CD47 are elevated in SARS-CoV-2-infected Caco-2 cells, Calu-3 cells, and air−liquid interface cultures of primary human bronchial epithelial cells. CD47 exerts an immunosuppressive activity via interaction with SIRPα in immune cells and as a thrombospondin-1 receptor [22,23]. In this context, human CD47 expression is discussed as a strategy to enable the xenotransplantation of organs from pigs to humans [69,70]. Moreover, a high CD47 expression is an immune escape mechanism observed in cancer cells, and anti-CD47 antibodies are under investigation as cancer immunotherapeutics [23,71]. Due its immunosuppressive action, CD47 expression is also discussed as a target for the treatment of viral and bacterial pathogens, including SARS-CoV-2 [22,24,72]. It has been demonstrated that cells infected with different viruses display enhanced CD47 levels, which function as a "do not eat me" signal, which interferes with the immune recognition of virus-infected cells [24]. Thus, our data indicating increased CD47 levels in a range of SARS-CoV-2 infection models and clinical samples further support the potential role of CD47 as a drug target for the mediation of a more effective antiviral immune response.
Moreover, we found that, although SARS-CoV-2 did not replicate in primary human monocytes, it increased the levels of the CD47 binding partner SIRPα in these cells. Hence, SARS-CoV-2 infection may affect the immune recognition of SARS-CoV-2-infected cells by upregulating both players of the CD47/SIRPα axis. Notably, other viruses and bacteria have previously been described to increase host cell SIRPα levels [73,74]. Moreover, elevated SIRP-α expression was recently reported in blood mononuclear cells of COVID-19 patients [75], and the CD47/SIRP-α interaction was associated with lung damage in severe COVID-19 [76].
High SARS-CoV-2 loads are associated with more severe COVID-19 and a higher risk of patient death [77,78]. Therefore, high CD47 and/or SIRPα levels may affect initial virus control resulting in enhanced virus levels, which may eventually lead to the hyperinflammation and immunopathology observed in severe COVID-19. Moreover, innate immune responses appear to be critically involved in the early control of SARS-CoV-2, and the deregulation of monocytes and macrophages seems to be a factor contributing to severe COVID-19 [79,80].
High CD47 levels have also been reported as a consequence of hyperglycemia and diabetes, which may contribute to the high risk of severe COVID-19 in diabetic patients [4]. Although there is no known direct impact of obesity on CD47 levels, obesity is associated with an increased risk of diabetes and other ageing-related conditions such as hypertension, which may result in elevated COVID-19 vulnerability [4].

Conclusions
Severe COVID-19 disease is a consequence of hyperinflammation ("cytokine storm") in response to SARS-CoV-2 infection [1]. Hence, the optimal time window for antiviral intervention is as early as possible to prevent disease progression to severe stages driven by immunopathology [1]. As the vast majority of cases are mild or even asymptomatic [1], an improved understanding of the processes underlying severe COVID-19 is required for the early identification of patients at high risk.
Here, we investigated a potential role of CD47 expression in determining COVID-19 severity. SARS-CoV-2 infection resulted in an enhanced expression of CD47 in different cell types. CD47 interferes with the host immune response by mechanisms including binding to SIRPα on immune cells. Notably, SARS-CoV-2 also increased SIRPα levels on primary human monocytes, indicating that SARS-CoV-2 can interfere with the immune response by elevating both binding partners of the CD47/ SIRPα axis.
Moreover, CD47 levels are elevated in groups at high risk for COVID-19, such as older individuals and individuals with hypertension and/or diabetes. Thus, high CD47 levels may predispose these groups to severe COVID-19. Additionally, CD47 is a potential therapeutic target that can be addressed with antibodies and small molecules [22][23][24]72]. Notably, targeting SIRPα also represents a therapeutic option that may be more specific, as SIRPα is restricted to monocytes and macrophages [81]. Further research will be needed to define the roles of CD47 and/or SIRPalpha in COVID-19 in more detail. Thus, our findings should also encourage other research groups to consider the potential relevance of these molecules in their COVID-19 research.

Conflicts of Interest:
The authors declare no conflict of interest.