key: cord-310928-g553afo9
authors: Murch, Simon H
title: Common determinants of severe Covid-19 infection are explicable by SARS-CoV-2 secreted glycoprotein interaction with the CD33-related Siglecs, Siglec-3 and Siglec-5/14
date: 2020-08-07
journal: Med Hypotheses
DOI: 10.1016/j.mehy.2020.110168
sha: 
doc_id: 310928
cord_uid: g553afo9

SARS-CoV-2 interaction with the ACE-2 receptor cannot alone explain the demography and remarkable variation in clinical progression of Covid-19 infection. Unlike SARS-CoV, the cause of SARS, several SARS-CoV-2 spike glycans contain sialic acid residues. In contrast to the SARS secreted glycoprotein (SGP), SARS-CoV-2 SGP are thus potential ligands for Sialic acid-binding Siglecs on host immune cells, known to regulate immune function. Such SARS-CoV-2 glycoproteins would contribute to immune deviation. CD33-related Siglecs are important immune regulators. Siglec-5 and -14 are paired receptors with opposed actions on the NLRP3 inflammasome, which is critical in early viral clearance. SGP binding in persons of Siglec-14 null genotype (30-70% in Black, Asian and Minority Ethnic (BAME) persons, 10% in North Europeans) would induce unopposed inhibitory signalling, causing viral persistence through inflammasome inhibition. Siglec-3 (CD33) and Siglec-5 are expressed on CD33 myeloid derived suppressor cells (CD33 MDSC). Immunosuppressive CD33 MDSC populations are increased in all groups at risk of severe Covid-19 infection. CD33 expression is increased in persons with the CD33 rs3865444 CC allele, associated with Alzheimer’s disease, who would thus show enhanced susceptibility. Viral SGP ligation of CD33, potentially in conjunction with Siglec-5, would promote expansion of CD33 MDSC cells, as occurs in cancers but at much greater scale. CD33 is expressed on CNS microglia, potentially activated by SGP penetration through the porous cribriform plate to cause anosmia. Genotyping of severe or fatal Covid-19 cases can confirm or refute this pathophysiological mechanism. Early data have confirmed extremely high-level increase of CD33 MDSC numbers in severe Covid-19 infection, consistent with the proposed mechanism.

An additional sialylated carbohydrate motif, associated with shorter glycan chain and less microheterogeneity, is found in sialyl-Tn glycans. Both CD33 and Siglec-5 are significantly bound by sialyl-Tn 6, 9 . Sialyl Tn glycan binding is a functionally important modulator of CD33 signalling, as identified for HIV envelope glycoprotein Gp120 3 .

SARS-CoV-2 has 16 n-linked glycans between the S1 subunit, which binds the angiotensin-2 receptor, and the S2 subunit, which mediates membrane fusion 10 . Glycosylation mapping has identified n-glycans terminating in 3'-SLacNAc structures at N-234 and N282 on spike-1 and N1098 on spike 2, while the O-glycan at T323 shows short sialic acid capped chains, including sialyl T antigen, and others trimmable to sialyl Tn 11, 12 . This represents a fundamental difference to SARS-CoV, where glycans show negligible sialylation.

The major SARS-CoV glycoprotein, mediating membrane fusion for viral entry in SARS, is also released into the circulation in soluble form 13 . Similar release through cleavage would allow SARS-CoV-2 sialylated secreted glycoproteins (SGP) to modulate immune response through CD33-r Siglecs. In particular, such interaction with Siglecs-3, -5 and -14 would explain specific features of the demography and pathogenesis of Covid-19 infection.

Siglec-5 and Siglec-14 are paired regulators of the Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing-3 (NLRP-3) inflammasome, which responds to danger signals in infection by production of inflammatory mediators (Figure 1 ) 14 . While unregulated NLRP-3 inflammasome activation may contribute to damaging inflammatory responses in chronic infection, it is critical in the early response to infection 6 necessary for viral eradication: viruses including Epstein-Barr virus and Zika virus have developed immune evasion strategies by inhibiting the NLRP3 inflammasome 14, 15 .

Siglec-5 and -14 are paired receptors, having essentially identical extracellular binding domains but differing intracellular signalling pathways 8 . Siglec-5 associates with tyrosine phosphatase SHP-1 to send an inhibitory signal upon ligation, while Siglec-14 associates with the adapter protein DAP-12 and the tyrosine kinase Syk to send an activating signal. Thus Siglec-5 binding downregulates NLRP3 inflammasome activity, while Siglec-14 binding upregulates it 8, 16 . Importantly, there is a common polymorphism, in which Siglec-14 is replaced by a Siglec-5 like fusion protein (Siglec 14/5) signalling via the inhibitory SHP-1 pathway identically to Siglec-5 17 . Individuals can express both Siglec-5 and -14 (Siglec-14 +/+ ), can have one allele where Siglec-14 is replaced by 14/5 (Siglec-14 +/heterozygous) or have both alleles where Siglec-14 is replaced by 14/5 (Siglec-14 -/-, known as Siglec-14 Null allele) 18 .

Thus, persons with Siglec-14 Null allele receive an inhibitory input to the NLRP3 inflammasome upon Siglec-5 binding while those without have a stimulatory input, greater in Siglec-14 +/+ homozygotes than heterozygotes ( Figure 1 ) 16, 18 . There are recognised functional consequences. Adults with the Siglec-14 null allele are at reduced risk for infective exacerbation of COPD 18 and may be less susceptible to severe tuberculosis 19 .

Conversely Siglec-14 null women are at increased risk of premature delivery If infected with GBS 17 .

There is substantial ethnic variation in Siglec-14 null allele, with a frequency of 50-70% in Chinese, South East Asians and Middle Eastern persons, 30-40% in Indo-Pakistanis and Sub-Saharan Africans and only 10% in Northern Europeans 9 . The pathogen drive currently or previously underlying such major variability remains so far unknown.

The NLRP3 inflammasome is a critical element in effective antiviral responses and its inhibition by pathogens an important cause of immune evasion, as seen in Zika virus infection 14, 15 . Binding of SARS-CoV-2 SGP to Siglec-5 would allow such immune evasion in persons of Siglec-14 null genotype through inflammasome inhibition. Newly recruited monocytes would be susceptible to similar inflammasome inhibition. Siglec-14 null women would also be more prone to preterm labour with Covid-19, as occurs if GBS colonised 8 .

This mechanism would put persons of origin from China or the Far East, the Middle East, the Indian subcontinent and Sub-Saharan Africa at increased risk of inability to clear SARS-CoV-2 virus upon initial exposure and thus of more severe and chronic disease. CD33 + MDSC populations release Arginase-1 to cause arginine depletion, inducing decreased T cell receptor (TCR)- chain expression and impaired adaptive immune responses 20, 21 . They also release the immunosuppressive cytokines TGF- and IL-10, as well as effector molecules such as nitric oxide and reactive oxygen metabolites. In addition B cell proliferation and antibody production are diminished by MDSC activation 23 .

CD33 MDSC populations are not restricted to cancer, and are increased in a number of other conditions. Indeed, in all other conditions known to be associated with increased Covid-19 mortality, circulating CD33 + MDSC are increased. Significant increase is seen in persons aged 61-76, compared to those aged 19-59, with further increase in frail elderly 22 .

Increased CD33+ MDSC numbers in cancer are associated with decreased TCR- expression, contributing to compromised anti-tumour responses 24 . CD33 + HLA-DR low/-MDSC cells are significantly increased in obesity, associated with decreased TCR- expression 25 . Type-2 diabetes mellitus and arterial hypertension cause increased circulating CD33 + MDSC cells producing immunosuppressive TGF- and IL-10 26 . Circulating CD33 + HLA-DR -MDSC are upregulated in smokers with or without chronic obstructive pulmonary disease (COPD) 27 .

In COPD this persists after stopping smoking, associated with significant downregulation of TCR- expression 27 . Stopping smoking reduces CD33+ MDSC numbers.

Persons with dementia, likely to have high age-related CD33 MDSC, have increased frequency of the CD33 rs3865444 CC allele which upregulates CD33 expression on microglia and immune cells 7 . Monocyte expression of CD33 is upregulated 7-fold in young persons carrying this allele, which explains over 70% of variance in CD33 cell expression 28 . While its putative role in dementia is that this CD33 variant impairs protective uptake of -amyloid, the relevance of this allele in CD33 rs3865444 CC carriers of whatever age in Covid-19

infection would likely be of increased response by MDSC and monocytes to a CD33-binding viral SGP. This may therefore be a second genetic predisposition to severe Covid-19

infection.

The excess of CD33 MDSC in persons with older age or frailty, diabetes, hypertension, COPD, cancer or previous malignancy or in those who smoke cigarettes would put them at There is evidence that persistent elevation of monocytic MDSC cells, identified as CD14 + HLA-DR low , is strongly associated with worse outcome in septic shock 29 . CD14 + HLA-DR low cells have been identified at high frequency in cases of severe Covid-19 infection with immune dysregulation 30 . Further characterisation would be required to determine how many of these cells are monocytic MDSC and how many are monocytes in which HLA-DR has been downregulated as suggested by the authors.

In addition, there is evidence that MDSC of proinflammatory potential can be released from the bone marrow in inflammation 31 . There is so far no clear consensus on their eventual phenotype but these new marrow derived cells are initially characterised as Early stage MDSC (eMDSC), which do not initially express lineage markers but do express CD33 and would thus be susceptible to SARS-CoV-2 SGP manipulation until the virus was cleared ( Figure 3 ). Proinflammatory MDSC are recognised in murine and human arthritis and in IBD,

where they suppress T cell proliferation, as do mature MDSC, but also strongly promote Th17 responses 31, 32, 33 .

Analysis of immune responses in severe Covid-19 infection identified two distinct groups 30 .

Around a quarter developed a macrophage activation syndrome with hypercytokinaemia and hyperferritinaemia in association with reduced total neutrophils and monocytes compared to persons with milder disease. Three quarters showed preservation of circulating cell numbers, with a state of chronic immune deviation but without macrophage activation syndrome. This association of reduced circulating cells with a hyperinflammatory response is consistent with bone marrow suppression, where release of new MDSC is prevented. By contrast, the prolonged state of immune suppression with immunopathology is explicable by viral SGP stimulation of the newly released proinflammatory eMDSC ( Figure 3 ).

There is increasing evidence that SARS-CoV-2 may invade the central nervous system 34 .

This would be most likely in severe disease with blood brain barrier disruption, and SARS-CoV-2 SGP would show tropism for CD33-expressing microglia.

A common presentation of Covid-19 disease is anosmia (loss of smell) with ageusia (loss of taste}. It has been postulated that the virus might gain access through the sieve-like barrier at the cribriform plate, in proximity to the olfactory bulb 34 . Penetration of a CD33 binding SGP would potentially induce activation of CD33-expressing microglia within the olfactory bulb to cause this symptom. This would not require ingress of whole viral structures and could occur even if blood brain barrier function was intact elsewhere.

In severe Covid-19 infection there is a significant increase of venous and arterial thrombosis 2 . Several mechanisms are likely to contribute to this. A CD33-r Siglec binding viral SGP would provide a further and independent risk factor for vascular thrombosis.

Analysis of 274 circulating cytokines in diabetic patients with critical limb ischaemia identified soluble Siglec-5 as the outstandingly increased marker, and confirmed this in a large second cohort, which excluded confounding risk factors 35 . Soluble Siglec-5 concentrations were considerably more than an order of magnitude greater than reported previously in healthy controls and patients with SLE 36 . The source was identified as irregularly-shaped Siglec-5+ cells that were present within atherosclerotic plaques but not normal vascular endothelium 35 . These cells were not further characterised, and thus may have been either Siglec-5+ macrophages or Siglec-5+ MDSC. The mechanism by which Siglec-5 contributes to the progression of vascular ischaemia has yet to be determined, but there is clear potential for modulation of the Siglec-5+ cells within existing vascular atherosclerotic plaques by such a Siglec-5 binding viral SGP.

There has been recent recognition of a Covid-19 associated syndrome of multisystem inflammation in previously healthy paediatric patients, usually without the initial presentation with respiratory disease seen in adults. By contrast, gastrointestinal symptoms were noted in around 90% of cases in both the UK and USA multicentre cohorts 37, 38 . What is notable about paediatric cases of Covid-19 infection is that SARS-CoV-2 is more readily detected in faeces than the respiratory tract 39 . There is now evidence that the virus can productively infect enterocytes, which strongly express the ACE-2 receptor. 40 . I suggest that the lack of respiratory symptoms at presentation may point to preferential infection via the gastrointestinal tract in children. The subsequent mechanism of disease would then be as for complicated adult disease (Figures 1-3) , but without the dominance of respiratory symptoms seen in adult disease.

In support of this, there was evidence of ethnic predisposition in both cohorts. The UK patients showed significant overrepresentation of Afro-Caribbean and Asian children (47% and 28% of the cohort respectively, compared to 8% and 7% of the overall UK population) 37 . The large USA cohort had ethnicity data on 147 children, of whom only 35 were White, non-Hispanic 38 . While most children had no recognised comorbidities, 37% of the US children for whom data was available had diagnosed or BMI-based obesity while the UK group showed a significant increase in the observed to expected weight ratio. Thus, a significant comorbidity in children may be an increase in MDSC cells due to overweight. It is also notable that MDSC populations are increased in children with atopic rhinitis 41 and asthma 42 .

The relative susceptibility of males is not explained by this, and the mechanism is more likely to relate to a distinct antiviral immune response (Figure 1 ). TLR7 responds to intracellular viral RNA in innate immune cells, and plasmacytoid dendritic cells of females produce more antiviral type-1 interferons than do males 42 , related to the number of X chromosomes and serum testosterone production 44 . This may underpin sex-related symptomatic differences in RNA viral infections more generally, but may have a fundamentally more important impact with a virus as virulent as SARS-CoV-2.

This model of SARS-CoV-2 pathogenesis can rapidly be supported or excluded. Such interaction could be confirmed or excluded by genetic analysis of persons with severe or fatal illness compared to the less affected.

1. Persons with homozygous Siglec-14 null allele would have higher incidence of severe disease than those Siglec-5 + Siglec 14 + , with heterozygotes intermediate. Preterm delivery would be more common in Siglec-14 null women, as in GBS infection.

2. Persons of CD33 rs3865444 CC allele, when stratified by disease susceptibility, would have higher incidence of severe disease than those with CD33 rs3865444 TT allele.

3. Increased numbers of CD33 MDSC at presentation in those who go on to develop severe disease. 

Proceeding from the direction of assessing in vitro SGP binding to cell lines or tissues suffers from the disadvantage that glycan expression is known to be modulated by enzymes within the Golgi apparatus of host cells during viral replication 12 . This may lead to impaired glycan maturation resulting in shorter glycan chains 12 . Thus, predicted glycans may not be representative of those functioning in vivo to mediate pathogenesis. However, detection of the footprints of Siglec-defined immunopathology would provide imperative for such extensive studies to be undertaken in order to characterise binding sites.

Prediction 4 appears to have been fulfilled, as MDSC populations (CD33+ but including both CD14+ monocytic and CD15+ granulocytic MDSC) were increased from 0.3% (IQR 0. 13-2.13) in healthy donors to 47.5% (IQR 28.4-65.6%) in Covid-19 infection 45 47 . This is a strong pointer towards a specific mechanism of induction, unique to Covid-19 infection. Prediction 6 is supported by evidence that proinflammatory MDSC of immature phenotype occur in human and murine arthritis, and strongly promote Th17 type responses 31, 32 . The cytokine storm in severe Covid-19 infection has indeed been characterised to be of Th17 type 48 , with over 30% of total CD4 cells being of Th17 phenotype (CD4 + CCR6 + ) in one wellstudied case with fatal outcome 49 .

If predictions based on this model are confirmed, inhibition of SARS-CoV-2 secreted glycoprotein interaction with host CD33-related Siglecs should be of clinical benefit. If the testing predictions are confirmed, the next stage would be to characterise the binding, which may be mediated by a single SGP or by two.

If the CD33-related Siglec-binding SGP is of sialyl Tn type, there may well be an existing sialyl Tn monoclonal that serendipitously blocks SGP binding to Siglecs. This would potentially be therapeutic.

If not, characterisation of SGP-Siglec binding would allow design of a small molecule that blocks interaction (possibly two), which would then be suitable for creating blocking immunoglobulin-(Ig)-fusion protein(s) for clinical use. In due course, this might allow persons who fail to respond to immunisation to be pre-treated to block Siglec binding sites, allowing them to be exposed to small infecting doses of SARS-Cov-2 in clinically controlled circumstances and thus generate a broad immune response, including to the pathogenic glycan determinants

The clinical course of Covid-19 infection is highly variable. The currently enigmatic determinants of disease severity can be fully explained on the basis of viral manipulation of host CD33-related Siglecs. There is evidence that both CD33 and Siglec-5 (thus also Siglec- 

The initial immune response to SARS-CoV-2 by innate immune cells, after its entry via the ACE-2 receptor on lung or gut epithelium, determines whether or not infection is swiftly controlled and minimally symptomatic. Such inflammasome activation is critical in an effective antiviral response. 5. NLRP3 activation is regulated by signals from cell surface Siglecs, if these are stimulated by Siglec ligands. An appropriately configured sialyl Tn or 3'SLacNac secreted glycoprotein would bind equally to Siglec-5 or Siglec-14 as their extracellular domain is almost identical. 6 . In a homozygous Siglec-14 positive person such glycoprotein binding induces strong activation of the NLRP3 inflammasome, while in a Siglec-14 null person this induces strong inhibition. In a person who is heterozygous, a more modest activation response occurs.

Thus, an infected person who is homozygous Siglec-14 positive is more likely to mount a successful early antiviral response than one who is Siglec-14 null, with heterozygotes making an intermediate response. Release of Arginase-1 by these cells, causing depletion of arginine, would synergise with release of the immunosuppressive cytokines IL-10 and TGF- to impair T cell proliferation and memory cell generation, as well as prevent the effective T-B cell interaction required for antiviral antibody production. B cell proliferation and antibody production would also be impaired. Release of nitric oxide and reactive oxygen species by these cells would contribute to ongoing inflammatory responses. 

The response by bone marrow as infection progresses may determine the clinical phenotype.

Persons suffering bone marrow suppression with severe disease will not release new MDSC into the circulation. These persons will show reduced numbers of circulating monocytes and neutrophils and will tend to progress towards macrophage activation syndrome as MDSC suppression wanes.

Those without bone marrow suppression maintain higher numbers of circulating cells. These include early phase MDSC, which can be stimulated through CD33 and Siglec-5. These cells are immunosuppressive but also proinflammatory, particularly promoting Th17 responses.

In both groups, a separate prothrombotic pathway will be driven by SGP stimulation of Siglec-5+ cells within atherosclerotic plaques. 

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