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Published in final edited form as: Antiviral Res. 2021 Dec 17;197:105226. doi: 10.1016/j.antiviral.2021.105226

CD47 expression attenuates Ebola virus-induced immunopathology in mice

Deepashri Rao 1, Kyle L O’Donnell 2, Aaron Carmody 3, Irving L Weissman 4, Kim J Hasenkrug 1,*, Andrea Marzi 2,*
PMCID: PMC8748401  NIHMSID: NIHMS1768306  PMID: 34923028

Abstract

It has been shown that a very early cell-intrinsic response to infection is the upregulation of CD47 cell surface expression, a molecule known for delivering a “don’t eat me signal” that inhibits macrophage-mediated phagocytosis and antigen presentation. Thus, blockade of CD47 signaling during lymphocytic choriomenigitis virus infections of mice has been shown to enhance the kinetics and potency of immune responses, thereby producing faster recovery. It seems counterintuitive that one of the earliest responses to infection would be immunoinhibitory, but it has been hypothesized that CD47 induction acts as an innate immune system checkpoint to prevent immune overactivation and immunopathogenic responses during certain infections. In the current study we examined the effect of CD47 blockade on lethal Ebola virus infection of mice. At 6 days post-infection, CD47 blockade was associated with significantly increased activation of B cells along with increases in recently cytolytic CD8+ T cells. However, the anti-CD47-treated mice exhibited increased weight loss, higher virus titers, and succumbed more rapidly. The anti-CD47-treated mice also had increased inflammatory cytokines in the plasma indicative of a “cytokine storm”. Thus, in the context of this rapid hemorrhagic disease, CD47 blockade indeed exacerbated immunopathology and disease severity.

Keywords: EBOV, filovirus, blocking antibody, cytokines, macrophages, dendritic cells, T cells

Introduction

CD47 is a ubiquitously expressed glycoprotein (Barclay and van den Berg, 2014; Liu et al., 2017) that binds to signal regulatory protein α (SIRPα or CD172a), which is expressed on macrophages, dendritic cells (DCs) (Barclay and van den Berg, 2014) and activated cytolytic T lymphocytes (Myers et al., 2019). The ligation of CD47 to SIRPα on macrophages and DCs phosphorylates immunoreceptor tyrosine-based inhibitory motifs (ITIMs) within the cytoplasmic tail of SIRPα resulting in an anti-phagocytic signal that regulates downstream immunoactivation pathways, usually in an inhibitory manner (Barclay and van den Berg, 2014). Such inhibitory signaling prevents the phagocytosis of normal, healthy cells. The loss of CD47-mediated inhibitory signaling from aged red blood cells is a primary signal leading to macrophage-mediated programmed cell removal (PrCR) (Bian et al., 2016). Evidence also indicates that CD47-SIRPα interactions are important contributors to the maintenance of peripheral tolerance via STAT3 phosphorylation and IL-10 expression (Toledano et al., 2013).

It has been shown that tumor cells upregulate CD47 to evade innate immune clearance (Betancur et al., 2017; Chao et al., 2011; Jaiswal et al., 2009; Majeti et al., 2009). Furthermore, blockade of CD47 with non-depleting antibody, or in conjunction with depleting antibodies such as rituximab, has been successfully used to eradicate tumors in both animal models (Chao et al., 2011, 2010; Jaiswal et al., 2009; Majeti et al., 2009; Schürch et al., 2019) and in clinical trials (Advani et al., 2018). The blockade of CD47-mediated inhibitory signaling allows positive signals such as those from damage associated molecular patterns to enhance phagocytosis by antigen presenting cells such as macrophages and dendritic cells, thereby directly eradicating tumor cells and also enhancing the cross-priming of T cells (Liu et al., 2015; Tseng et al., 2013). Interestingly, it was also recently shown that CD47 is upregulated in infected cells (Tal et al., 2020). CD47 upregulation is induced by stimulation of pathogen recognition receptors (PRRs) and it has been speculated that this brake may be important in preventing overinflammation during certain infections (Tal et al., 2020). However, this has not been shown for any infection. Interestingly, all pox viruses encode a CD47 mimic (Cameron et al., 2005a; Campbell et al., 1992). The CD47 mimic of Myxoma virus not only downregulates macrophage and T cell activation in vivo (Cameron et al., 2005a), but is a potent virulence factor required for in vivo virus spread (Cameron et al., 2005b). These findings suggest that antibody-mediated CD47 blockade during viral infection might generally enhance immunity and lead to quicker virus clearance. Indeed, a recent study showed that blockade of CD47 enhanced the activation of DCs, increased the kinetics and potency of CD8+ T cell responses and significantly improved control of lymphocytic choriomenigitis virus (LCMV) infections in mice (Cham et al., 2020). However, the effects of CD47 blockade might be dependent on the type of pathogen and the pathology and immune responses induced by that pathogen. In the current experiments, we investigated the effects of CD47 blockade during Ebola virus (EBOV) infections in mice. EBOV is a member of the Filoviridae family and causes sporadic hemorrhagic disease outbreaks in humans in Africa with case fatality rates up to 90% (Feldmann and Geisbert, 2011). DCs and macrophages have been described as early target cells of EBOV infection (Geisbert et al., 2003a, 1992), making the CD47 signal an interesting target for investigation. Furthermore, transcriptional data from collaborative cross mice indicated that early activation of macrophages and DCs was associated with resistance to EBOV infection (Price et al., 2020). For this study we used a well-established mouse model of infection with mouse-adapted EBOV (MA-EBOV) (Bray et al., 1999).

Materials and methods

Ethics statement

All infectious work was performed at the required containment level at the Integrated Research Facility, Rocky Mountain Laboratories (RML), Division of Intramural Research (DIR), National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH) according to standard operating protocols (SOPs) approved by the RML Institutional Biosafety Committee (IBC). The animal work was approved by the Institutional Animal Care and Use Committee (IACUC) (Animal study protocol #2020-030-E) and performed according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International and the Office of Laboratory Animal Welfare. All procedures on animals were carried out by trained and certified personnel following SOPs approved by the IBC. Humane endpoint criteria in compliance with IACUC-approved scoring parameters were used to determine when animals should be humanely euthanized. Any animal with weight loss equal to or greater than 20%, or signs of the following, was euthanized: ataxia, lethargy (animal is reluctant to move when prompted), bloody discharge from nose, mouth, rectum or urogenital area, tachypnea, dyspnea or paralysis of the limbs.

In vitro infection of RAW 264.7 cell line

The RAW 264.7 mouse macrophage cell line was obtained from ATCC. Cells were seeded at 4×104 cells/well in 48-well flat-bottom plates. When they were 70% confluent in log-phase of growth, cells were infected with 0.01, 0.1 or 1 multiplicity of infection (MOI) of MA-EBOV in triplicates and incubated at 37°C with 5% CO2. Cells were harvested at 24 and 48 hours post infection (hpi) and examined for expression of CD47 (miap301, BioLegend) or CD86 (GL1, BioLegend) by flow cytometry. Uninfected controls in duplicate were included for both time points analyzed.

Mouse infection and treatment

Experiments were conducted using female C56BL/6 mice obtained from NIAID/Taconic and bred at the RML. The mouse model for EBOV using MA-EBOV has been established and used at the RML using only female mice (Bray et al., 1999; Haddock et al., 2018). Therefore, only female mice were used in this study. All of the mice were 12 to 20 weeks old at the beginning of the experiment. Mice were kept naïve or infected with 10 FFU of MA-EBOV (Bray et al., 1999) in 0.2mL of sterile DMEM via the intraperitoneal (IP) route. For treatment, mice were IP-injected with either 100μg of anti-CD47 antibody (BioXCell) or isotype control antibody (BioXCell). Treatment began 3 hpi and was repeated daily up to and including day 5. Mice were anesthetized (inhalational isoflurane) prior to infection and treatment with blocking antibodies. Treatment on day 6 was not carried out as the mice had reached endpoint criteria and were euthanized. At the time of euthanasia, spleens and if possible, a terminal blood sample were collected by cardiac puncture and divided between EDTA and serum separator tube for further analysis. Virus titers were determined as previously described (Marzi et al., 2018).

Surface and intracellular antibody staining for flow cytometry

The harvested spleens were mashed and single-cell suspensions of the spleens were stained with the following surface antibodies: anti-CD45 (30-F11, BD Biosciences), LIVE/DEAD Fixable Blue cell stain (Invitrogen), anti-CD19 (ID3, BD Biosciences), anti-CD8a (53-6.7, BioLegend), anti-CD27 (LG7F9, eBioscience), anti-CD11b (M1/70, BioLegend), anti-CD11c (HL3, BD Biosciences), anti-CD86 (GL1, BioLegend), anti-NK1.1 (PK136, BioLegend), anti-NKp46 (29A1.4, BioLegend), anti-MHC Class II (M5/114.15.2, BD Biosciences), anti-F4/80 (BM8, BioLegend), anti-CD80 (16-10A1), anti-CD3 (17A2, BioLegend), anti-CD4 (RM4-5, Invitrogen), anti-CTLA-4 (UC10-4B9, BioLegend), anti-PD-1 (29F.1A12, eBioscience), anti-CD28 (37.51, BioLegend), anti-CD44 (IM7, BD Biosciences), anti-CD43 (1B11, BioLegend), anti-CD47 (miap301, BioLegend), anti-CD62L (MEL-14, BioLegend), anti-CD25 (PC61.5, eBioscience), and anti-CD107a (1D4B, BioLegend). Intracellular staining was then performed with a FOXP3 permeabilization and fixation kit (eBioscience) according to manufacturer’s recommendations using the following antibodies: Ki-67 (B56; BD Biosciences) and GrB (QA16A02, BioLegend). Flow cytometric data were collected with a Beckman Coulter Cytoflex LX (6-L NUV) flow cytometer and analyzed using FlowJo software (Tree Star).

Analysis of serum cytokines

Infected mouse sera were inactivated by γ-irradiation (4 MRad) and removed from the maximum containment laboratory according to SOPs approved by the RML IBC. Serum from naïve and MA-EBOV infected mice treated with anti-CD47 or isotype-control was analyzed for cytokines using the Bio-Plex Pro Mouse Cytokine 23-plex Assay (Bio-Rad Laboratories, Inc.) following the recommendations of the manufacturer. The samples were analyzed on a Bio-Plex 200 system.

Statistical analysis

Differences in viral loads and body weights were analyzed by Mann-Whitney test. Mantel-Cox test was used to analyze difference in survival of mice. Percentages of immune cell populations and increases in expression of specific cell markers between groups were compared by Mann-Whitney test or Kruskal-Wallis test with Dunn’s multiple comparisons. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All statistical analyses were performed using Prism version 9 (GraphPad).

Results

Infection with MA-EBOV upregulates CD47 and CD86 in vitro

To determine whether EBOV infection induced upregulation of CD47 cell surface expression, as has been shown for numerous other pathogens (Tal et al., 2020), we infected the mouse macrophage cell line RAW 264.7 with MA-EBOV and analyzed CD47 expression 24 and 48 hours later. Cells infected at the highest MOI (MOI 1) showed the most increased expression of CD47 at 24 hpi compared to uninfected cells (Figure 1A). By 48 hours, expression was increased at all MOIs tested with MOI 1 being highest and statistically significant (Figure 1B). Macrophages are primary targets for EBOV and infection has been shown to increase macrophage activation, which plays a significant role in disease pathogenesis (Bray and Geisbert, 2005; Escudero-Pérez et al., 2014; Geisbert et al., 2003a; Rogers and Maury, 2018). Therefore, we also analyzed the effect of EBOV infection on the activation (CD86 expression) of RAW 264.7 cells. Already at 24 hpi, CD86 cell surface expression was increased at all MOIs tested (Figure 1 C). Thus, EBOV infection increased the expression of both CD47 and CD86 in RAW 264.7 macrophage cells.

Figure 1. CD47 CD86 cell surface expression on RAW 264.7 cells after MA-EBOV infection.

Figure 1.

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RAW 264.7 cells were infected with MA-EBOV at a MOI of 0.01, 0.1 or 1 or left uninfected. Cells were harvested and analyzed for upregulation of CD47 at (A) 24 and (B) 48 hpi or (C) CD86 at 24 hpi compared to naïve controls. Statistically significant results as determined by Kruskal-Wallis test are indicated as follows: *p<0.05. hpi hours post-infection.

Anti-CD47 antibody therapy increases viral load in MA-EBOV infection

MA-EBOV infection of mice causes lethal disease characterized by rapid virus replication with extensive spread to organs including the liver and spleen as early as 2 days post-infection (dpi)(Bray et al., 1999). To determine if a post-exposure prophylactic treatment of CD47 blockade would be effective in controlling virus spread and pathology, we began anti-CD47 antibody treatment 3 hpi. Daily therapy was continued through day 5. At 6 dpi, the mice were euthanized, and the organs were harvested for analyses. An infectious virus assay was performed to determine EBOV titers in blood, liver and spleen samples from mice treated with anti-CD47 or isotype control. The results showed similar levels of infection in the blood and liver, but significantly higher EBOV titers in the spleens of anti-CD47 treated mice compared to controls was observed (Figure 2A).

Figure 2. Effect of anti-CD47 therapy on infectious virus titers, body weight loss and survival of MA-EBOV-infected mice.

Figure 2.

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C57BL/6 mice were infected with 10 FFU MA-EBOV and treated with either 100 μg of anti-CD47 or an isotype antibody. Treatment occurred by intraperitoneal injection beginning at 3 hours post-infection with repeated daily doses until day 5. (A) Infectious virus titers were analyzed in the blood, liver and spleen at 6 days post-infection (n=6 mice per group). Data are shown as geometric mean ± geometric SD. Additional groups of 8 mice were monitored daily for changes in body (B) weight and (C) survival. Mann-Whitney test (A, B) and Mantel-Cox test (C) were used to analyze statistical differences between treated and control mice. Statistically significant results are indicated as follows: *p<0.05, **p<0.01, and ***p<0.001.

A primary clinical sign of MA-EBOV infection is loss of body weight, followed by rapid progression of hemorrhagic disease (Haddock et al., 2018). Analysis of body weights showed that anti-CD47-treated mice had significantly increased loss of body weight at 4 and 5 dpi (Figure 2B). By 6 dpi, all eight mice in the anti-CD47-treated group and two of eight mice in the isotype-control-treated group developed clinical signs that met humane end-point criteria and were euthanized. The remaining 6 mice in the control group met end-point criteria on the following day. This one day reduced survival in the treated mice was statistically significant (p=0.003; Figure 2C). Thus, the anti-CD47-treated mice had increased virus loads, increased weight loss and more rapidly progressing disease resulting in reduced time to death. Since delivery of anti-CD47 antibody starting at 3 hpi with MA-EBOV did not disrupt the usual development of pathology and instead made the clinal signs worse, timepoints earlier than 6 dpi were not investigated.

Anti-CD47 and MA-EBOV effects on Antigen Presenting Cells

To analyze whether CD47 blockade and/or MA-EBOV infection induced the activation of macrophages in vivo, we used flow cytometry to detect CD86. The gating strategies for the flow cytometry analyses are shown in Figure S1. At 6 days post-treatment with anti-CD47, splenic antigen presenting cell (APC) subsets from uninfected mice did not show an increased expression of CD86 compared to isotype controls (Figure 3A–D). All APCs from MA-EBOV-infected mice had extremely high levels of CD86 expression (Figure 3E–H). CD47 blockade appeared to increase mean CD86 expression levels but the difference did not quite reach the accepted level of statistical significance in the macrophages (p=0.0649) (Figure 3E).

Figure 3. Effect of CD47 blockade on activation of macrophages and dendritic cells in MA-EBOV infection.

Figure 3.

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The expression of CD86 on splenic macrophages (A, B) and dendritic cells (B-D, F-H) was analyzed from naïve (top panel) and MA-EBOV-infected (bottom panel) mice treated with either IgG1 isotype or anti-CD47 6 days post-infection. To determine if CD86 upregulation was specific to DC subsets, both CD11b+ DCs in B, F and CD8+ DCs in C, G as well as plasmacytoid DCs (pDCs) (CD8− F4/80− CD11b− CD11c+ in D, H) were examined. n=6 mice per group. Data are shown as mean ± SEM. Results were not statistically significant by Mann-Whitney test, though upregulation of CD86 in macrophages was marginal with a P value of 0.0649.

CD47 blockade results in reduced frequency of B cells and CD8+ T cells in MA-EBOV-infected mice

Lymphopenia of both B and T cell subsets in the spleen, lymph node and thymus in MA-EBOV infections has been previously described (Bradfute et al., 2007; Geisbert et al., 2000). In addition, high activation of lymphocytes has also been reported in human EBOV infection (McElroy et al., 2015). To determine whether CD47 blockade affected lymphopenia or activation in lymphocytes during MA-EBOV infection, cell subsets were analyzed by flow cytometry at 6 dpi when mice reached endpoint criteria and were euthanized. Both naïve and MA-EBOV-infected mice treated with anti-CD47 had a reduced proportion of B cells (CD19+) compared to the mock-treated controls (Figure 4A). Although reduced in proportions, the B cells in the anti-CD47 treated MA-EBOV-infected mice had significantly higher expression of MHC Class II (Figure 4B), indicative of activation. The proportions of CD3+ T cells in naïve but not EBOV-infected mice treated with anti-CD47 were significantly reduced compared isotype-treated controls (Figure 4C). In contrast to the B cells, the proportions of CD4+ T cells of both naive and infected mice treated with anti-CD47 were not significantly different than controls (Figure 4D). Within the CD4+ helper T cell subset there was no difference in the level of activation (CD43+) between treated and control groups in both naïve and EBOV-infected mice (Figure 4E), however, MA-EBOV-infected mice had significantly higher levels compared to naïve control-treated mice (Figure 4E). There was however, a significant increase in the level of expression of the inhibitory molecule, CTLA-4 on CD4+ T cells in EBOV-infected mice receiving anti-CD47 therapy (Figure 4F), the high expression of which has previously been shown on T cells in fatal human cases of Ebola virus disease (EBOD) (Ruibal et al., 2016). The proportion of CD4+ regulatory T cells (CD25+FoxP3+) in EBOV-infected mice, although significantly lower than naïve mice, did not show any difference with anti-CD47 treatment (Figure 4G).

Figure 4. Effect of CD47 blockade on B and T cells in MA-EBOV infection.

Figure 4.

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The effect of anti-CD47 therapy on splenic B cells (A, B), CD3+ T cells (C), CD4+ T cells (D-F), regulatory T cells (G) and CD8+ T cells (H-L) was analyzed on day 6 post-infection. CD45+ cells were analyzed for the percentage of splenic CD19+ B cells (A). B cells from EBOV-infected mice were analyzed for the expression of MHC Class II (B). Lymphocytes gated on live cells were analyzed for the percentage of CD3+ T cells (C). CD3+ T cells were analyzed for the frequency of CD4+ FoxP3- T cells (D). CD4+ T cells were further analyzed for the following: expression of CD43 (E), and MFI of CTLA-4 on CD4+ FoxP3- T cells in EBOV-infected mice (F). CD3+CD4+ T cells were analyzed for the percentage of CD25+FoxP3+ Treg cells (G). CD3+ T cells were also analyzed for the frequency of CD8+ T cells (H). CD8+ T cells were further analyzed for the following: expression of CD43 (I), CTLA-4 (J), MFI of CTLA-4 on CD8+ T cells in EBOV-infected mice (K), and surface expression of CD107a (L). n=6 mice per group. Data are shown as mean ± SEM. Data comparing the frequency of cells was analyzed by Kruskal-Wallis test and MFIs were compared using Mann-Whitney test. Statistically significant results are indicated as follows: *p<0.05, **p<0.01, and ***p<0.001.

Blockade of CD47 resulted in a significant decrease in the proportion of splenic CD8+ T cells in EBOV infection compared to naïve mice (Figure 4H). EBOV infection led to an increase in the level of activation (CD43+) but activation was not altered by anti-CD47 treatment (Figure 4I). EBOV infection also resulted in a significantly higher percentage of CD8+ T cells expressing CTLA-4 (Figure 4J). Furthermore, the expression levels of CTLA-4 were significantly higher in the anti-CD47-treated mice compared to isotype control-treated mice (Figure 4K). Regardless of infection, anti-CD47 was also associated with a significant increase in the proportion of CD8+ T cells expressing CD107a (Figure 4L), a surrogate marker for recent cytolytic activity.

Increased levels of proinflammatory cytokines with anti-CD47 therapy

Production of inflammatory cytokines has been reported in EBOV infections of humans, NHPs and mice (Baize et al., 2002, 1999; Bird et al., 2015; Bradfute et al., 2012; Bradfute and Bavari, 2011; Villinger et al., 1999; Wauquier et al., 2010a) and a proinflammatory “cytokine storm” has been associated with higher mortality in EBOV infections (Geisbert et al., 2003a; Misasi and Sullivan, 2014; Wauquier et al., 2010b). In naïve mice at 6 days post-treatment with anti-CD47, there were no significant differences in the levels of the cytokines/chemokines analyzed compared to isotype control (Figure 5 A–J). To determine if anti-CD47 therapy may lead to increased inflammatory cytokine/chemokine responses in the context of MA-EBOV infection, serum cytokine/chemokine levels were examined at six days post-infection. In comparison to isotype-treated mice, numerous proinflammatory mediators that can be produced by macrophages were found to be significantly increased in the anti-CD47-treated mice including: IL-1β, IL-3, GM-CSF, CXCL1, IL-12 and TNF-α (Figure 5 K–P). In addition, several T cell-produced cytokines were also significantly higher in the anti-CD47-treated mice including: IL-2, which has pleiotropic effects on CD4+ Th1, Th2 and regulatory T cells as well as CD8+ T cells (Figure 5Q); the Th2 cytokines IL-5 and IL-13, and the proinflammatory Th17 cytokine, IL-17a (Figure 5 R–T). Thus, in the context of MA-EBOV infection anti-CD47 therapy was associated with hypercytokinemia and more severe pathology.

Figure 5. Effect of CD47 blockade on serum cytokines and chemokines in mice with and without MA-EBOV infection.

Figure 5.

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The effect of anti-CD47 therapy on the level of cytokines and chemokines in the serum of naïve (A-J) and MA-EBOV infected mice (K-T) was measured 6 days post-infection. The levels of IL-1β (A, K), IL-3 (B, L), GM-CSF (C, M), CXCL1 (D, N), IL-12 (E, O), TNF-α (F, P), IL-2 (G, Q), IL-5 (H, R), IL-13 (I, S) and IL-17A (J, T) are depicted as mean ± SEM picograms/milliliter. n=6 mice per group in the naïve mice (A-J). n=3 (anti-CD47 treated) and n=5 (isotype-control treated) mice in MA-EBOV infection (K-T). Statistically significant results determined by Mann-Whitney test are indicated as follows: *p<0.05. Dotted line indicates limit of detection.

Discussion

Immune responses perform the critical function of protection against pathogenic infections via the induction of extremely potent cellular and soluble effector mechanisms capable of inducing fever, inflammation, and cell death. Left uncontrolled, such mechanisms may cause immunopathology including tissue damage, multiorgan failure and even death of the host. Thus, complex immunoregulatory mechanisms including what have been termed “immune checkpoints” have evolved to protect the host from immunopathology during immune responses, even at the cost of slowing pathogen clearance or allowing the outgrowth of tumors. Hence, inhibitors of adaptive immunity checkpoints such as anti-CTLA-4, anti-PD-1 and anti-PDL-1 are at the forefront of cancer immunotherapies designed to enhance immune responses dampened by the host (Robert, 2020). Recent studies including the present one, demonstrate that the innate immune system also contains checkpoints, including CD47 expression. As such, CD47 blockade is currently showing promise for cancer therapy, especially when combined with anti-tumor monoclonal antibodies (Advani et al., 2018; Upton et al., 2021). As demonstrated by the results of the current study, the use of checkpoint inhibitors to potentiate immune responses to acute infections may pose a more difficult immunoregulation problem than treating cancers, especially for a virus such as EBOV that already induces a strong inflammatory response. On its own, CD47 blockade did not induce cytokine storm (Fig. 5A–J), but in the context of acute EBOV infection, inflammatory cytokine production was significantly increased and associated with more severe clinical signs and shorter time to death. This finding strongly supports the hypothesis (Tal et al., 2020) that pathogen-induced upregulation of CD47 is an immune checkpoint response, which has evolved to reduce immunopathology in the context of certain infectious diseases (Tal et al., 2020).

In a previous study, antibody-mediated blockade of CD47 signaling during LCMV infection produced significantly increased activation of DCs, faster expansion of functional CD8+ T cells, and improved virus control (Cham et al., 2020). Furthermore, it was shown that faster virus clearance was dependent on both the antigen presenting cells and the CD8+ T cells. When we used the same CD47-blocking antibody following infection with MA-EBOV, we found significantly more recently cytolytic (CD107a+) CD8+ T cells, but virus control was worse rather than better. Thus, it was of interest to determine why that happened and what the implications are for using CD47 blockade as a therapeutic for infectious diseases. The progression of disease in EBOV-infected mice is very rapid and lethal within a week. In such a rapid infection, it is critical that the innate response is able to limit virus replication and spread until the adaptive response can take over, and why we sought to enhance the innate response. Given that cytokine storm has been linked to lethal disease in both animal models and humans (Bird et al., 2015; Bradfute et al., 2012; Geisbert et al., 2003a; Misasi and Sullivan, 2014; Price et al., 2020; Wauquier et al., 2010a), it is likely that the increased inflammatory cytokine production observed with CD47 blockade contributed to faster EBOV lethality. Macrophages are highly susceptible to infection by EBOV (Bray and Geisbert, 2005; Escudero-Pérez et al., 2014; Geisbert et al., 2003; Rogers and Maury, 2018) and the glycoprotein (GP) shed from EBOV-infected cells binds to and activates uninfected macrophages and DCs to induce the secretion of inflammatory cytokines leading to immune activation and vascular permability (Escudero-Pérez et al., 2014). Consistent with those reports, we found very strong activation of macrophages in MA-EBOV infected mice, with more than forty fold increased mean expression levels of CD86 (compare panel A and E in Fig. 3). Anti-CD47 treatment resulted in higher mean expression of CD86, but there was some variability between animals and the difference did not quite reach statistical significance (P = 0.0649). It may be that CD47 blockade increased the activation of macrophages already overactivated by MA-EBOV infection to elicit the significantly exacerbated inflammatory cytokine response demonstrated in Figure 5, but further studies including depletion experiments would be required to definitively identify the cell types involved. In the context of such cytokine storm, it is unlikely that the slight increase in recently cytolytic CD8+ T cells we observed could have any major impact on disease control.

Exacerbation of disease in anti-CD47-treated mice was associated with increased viral loads in the spleen but not the blood or liver (Fig. 2A). It has been demonstrated that anti-CD47 antibodies can penetrate tissues in vivo (Gholamin et al., 2017; Sikic et al., 2019), although this has not been shown for spleen or liver. One obvious difference between these tissues is that the spleen contains lymphoid tissue with relatively abundant targets for EBOV replication such as APCs, including monocytes, whereas the liver does not. Although an ex vivo study demonstrated that blood monocytes do not support significant virus uptake (Martinez et al., 2013), more recent studies support a role for blood monocytes in EBOV infection (Lüdtke et al., 2016; Menicucci et al., 2017; Versteeg et al., 2017). Furthermore, EBOV infection of macrophages and/or monocytes results in a “cytokine storm” and contributes to pathology (Feldmann et al., 1996; Geisbert et al., 2003c, 2003b). That said, we cannot confirm at this point why we did not observe increased virus replication in blood or liver as was observed in the spleen.

The ability of CD47 blockade to activate the innate immune response in a pathogen-independent manner inclines it toward general applicability as an infectious disease therapeutic. However, the current study demonstrates that CD47 blockade can also thwart host mechansims needed to reduce immunopathogenesis. Thus, it is of extreme importance to understand the mechanisms of pathogenesis for specific pathogens to determine whether CD47 blockade is applicable. It would also be valuable to investigate earlier timepoints to understand the kinetics of the immune response and how blocking CD47 alters the course of the disease. Obviously, in vivo studies will play an integral role in determining the complex interactions between hosts, pathogens and potential therapeutics.

Supplementary Material

1

Acknowledgements

We thank Ronald Messer (NIAID, NIH) and the Rocky Mountain Veterinary Branch (NIAID, NIH) for assistance with this study. The study was funded by the Intramural Research Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Footnotes

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Conflict of interest

The authors have no conflict of interest.

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