The role of sialic acids in the immune recognition of xenografts

Abstract

Presentation of sialic acid (Sia) varies among different tissues and organs within each species, and between species. This diversity has biologically important consequences regarding the recognition of cells by “xeno” antibodies (Neu5Gc vs Neu5Ac). Sia also plays a central role in inflammation by influencing binding of the asialoglycoprotein receptor 1 (ASGR-1), Siglec-1 (Sialoadhesin), and cellular interactions mediated by the selectin, integrin, and galectin receptor families. This review will focus on what is known about basic Sia structure and function in association with xenotransplantation, how changes in sialylation may occur in this context (through desialylation or changes in sialyltransferases), and how this fundamental pathway modulates adhesive and cell activation pathways that appear to be particularly crucial to homeostasis and inflammation for xenografts.

1 ∣. SIALIC ACID STRUCTURE

Sialic acids (Sias) are negatively charged monosaccharides typically expressed on the terminating branches of N-glycans, O-glycans, and glycosphingolipids (gangliosides) on the surface of virtually all vertebrate cells. The Sia family contains more than 50 members, but two common “primary” Sias have been described as follows: 5-acetamido-2-ke to-3,5-dideoxy-D-glycero-D-galactonononic acid (N-acetylneuraminic acid, or Neu5Ac) and 2-keto-3-deoxy-D-glycero-D-galactonononic acid (2-keto-3-deoxynononic acid, or Kdn). All Sias contain a nine carbon backbone. Except for some bacterial Sia,¹ all other types of Sia are metabolically derived from these two precursors. Neu5Ac is more common than Kdn in most vertebrate cell types and is expressed by all mammals. In mammals, except humans, Neu5Ac is converted into N-glycolylneuraminic acid (Neu5Gc) by conversion of CMP-Neu5Ac to CMP-Neu5Gc in the cytoplasm by the CMP-Neu5Ac hydroxylase, which is encoded by the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene. During the evolution, the human CMAH gene became non-functional. Consequently, humans predominantly express Neu5Ac (and little to no Neu5Gc), whereas most other mammals predominantly express Neu5Gc. As Neu5Gc, like Gal α1,-3Gal, is expressed by gut bacteria and not by “self,” it is not surprising that a substantial fraction of anti-carbohydrate antibodies that bind to cells from candidate organ xenograft donors are directed against these two antigens.

There are additional layers of diversity in the structure of Sia related to their linkage to the underlying glycan chain. Sias are usually found at the non-reducing terminal position of glycoconjugate sugar chains, linked to a galactose (Gal), a β-D-N-acetylgalactosaminyl (GalNAc), or a β-D-N-acetylglucosaminyl (GlcNAc) residue. Sia bind to underlying residues via α-linkages between their second carbon (C-2) and either the third carbon (C-3) or sixth carbon (C-6) positions of Gal, GalNAc, or GlcNAc. When Sia are linked to other Sia residues at the eighth carbon (C-8), they are classified as poly-sialic acids.² Additional chemical modifications of Sias such as hydroxylation and acetylation have been described which contribute to the enormous variety of glycan structures on cell surfaces and contribute to the distinctive phenotype and physiologic diversity of different cell types.

2 ∣. SIALYLATION: A DYNAMIC BALANCE

The “net” cellular sialylation/desialylation state at any time depends on the combination of chemical synthesis of Sia-bearing proteins and lipids, the rate of transfer of Sia-expressing molecules to the cell surface, and the rate of loss of Sia residues. Loss of Sia from the cell surface may occur either by active removal (which is believed to be primarily enzymatic) or by shedding, which occurs mainly by release of Sia-bearing molecules from the cell surface. In eukaryotic cells, free Sia (derived from biosynthesis or recycled/recovered from the lysosome) is activated into the nucleotide donor CMP-Sia, by CMP-Sia synthases in the nucleus. The CMP-Sia products then return to the cytoplasm and are delivered into the lumen of Golgi compartments, where Sias are transferred onto newly synthesized glycoconjugates passing through Golgi compartments by the action of sialyltransferases (ST). Finally, during molecular turn-over and recycling of cell-surface molecules, glycoconjugates are desialylated in endosomal/lysosomal compartments and return to the Golgi to undergo re-sialylation.

Some mammalian cells also express sialidases on their cell surface (plasma membrane) conditionally after cell activation, which are preformed and mobilized from intercellular reservoirs such as endosomes. Cell-surface sialidases (neuraminidases) have been implicated in the rapid shedding of cell-surface Sias during activation of certain cell types (eg neutrophils). Altogether, the net amount of sialylation on a cell’s surface depends on the opposing activity of sialyltransferases, which promote sialylation, and sialidases, which strip Sia residues from the cell surface.

3 ∣. SIALYLTRANSFERASES

Human sialyltransferases (STs) are a family of at least 20 different intracellular, Golgi membrane-bound glycosyltransferases.³ They transfer Sia onto newly synthesized glycoconjugates during their passage through the Golgi.² STs are determined based on the acceptor structure on which they act, as well as on the sugar linkage they form. For example, a group of ST adds Sia with an α-2,3 linkage to galactose, resulting in Sia-α-2,3-Gal, while other ST add Sia with an α-2,6 linkage to galactose or N-acetylgalactosamine, resulting in Sia-α-2,6 Gal or Sia-α-2,6-GalNAc Specifically, STs are classified as ST3, ST6, or ST8 according to their formation of α-glycosidic bonds between the C2 of the Sia and the 3′-,6′-, or 8′-hydroxyl group of the acceptor, respectively. Each of the three ST families has up to six subfamilies that differ based on their substrate specificity. For example, ST6Gal-II is an oligosaccharide-specific enzyme toward oligosaccharides that have a Gal-β-1,4-GlcNAc sequence, whereas ST6Gal-I demonstrates broad substrate specificities.³ ST activity and expression vary upon cell activation, which in turn is influenced by both physiological and pathological processes. In mouse livers during inflammation, there was upregulated expression of β-galactoside α2,3-sialyltransferases (ST3Gal-I and ST3Gal-III) and β N-acetylgalactosaminide α2,6 sialyltransferase (ST6GalNAc-VI) as well as β-galactoside α2,6-sialyltransferase (ST6Gal-I) mRNAs. The endothelial surface also expresses ST activity.⁴ Most of the human and mouse ST family members’ sequences have been determined. In contrast, most pig ST genes have not been fully characterized, but this is being looked into.⁵

4 ∣. SIALIDASES

By convention, eukaryotic neuraminidases are called sialidases, reserving the term neuraminidase for other organisms. Sialidases remove the terminal Sia residues at the non-reducing end of glycoconjugates. There are four mammalian sialidase genes in eukaryotic organisms, which are classified as neuraminidase (NEU) 1-4. Neuraminidases are also expressed by human pathogens such as the influenza virus.

Sialidases are differentially expressed in various cells and tissues/organs, and in subcellular spaces, as detailed in the next three paragraphs.²⁶

NEU1 is localized in lysosomes, where it is associated with Cathepsin A and β-galactosidase (optimum pH 4.4-4.6). NEU2 is found in the cytosol (pH 6.0-6.5). NEU3 is associated with the plasma membrane (pH 4.5-4.7 and 6-6.5, depending on the changes occurring in the plasma membrane). NEU4 is found in lysosomes, mitochondria, or endoplasmic reticulum (pH 4.5-4.7).

Sialidases display different activities for various Sia linkages. NEU1, 2, and 4 prefer α2,3 linkages. NEU3 prefers α2,3 or α2,6 linkages.⁷ Each sialidase also targets different substrates: NEU1, 2, and 3 target oligosaccharides; NEU2 and 4 glycoproteins; NEU2, 3, and 4 gangliosides; and NEU1 glycopeptides. In human tissues, NEU1 generally shows the strongest expression by PCR and Western blot, 10-20 times greater than those of NEU3 and NEU4. The expression of NEU2 is extremely low in human cells.⁸ Sialidase expression and activity can be upregulated in response to inflammation both from increase of protein expression as well as translocation to different cellular compartments or to the surface of cells.

Sialidase expression is dynamically regulated in immunocytes. For example, in human T cells, both NEU1 and NEU3 mRNAs are induced by T-cell receptor stimulation. Several cytokines, including interleukin IL-2 and IL-13, are induced upon the upregulation of these sialidases.⁹ In addition to its intracellular location, NEU1 and NEU3 were found to translocate from lysosomes to the plasma membrane during immune activation or differentiation in several cell types, such as macrophages,¹⁰ endothelial cells,¹¹ and erythrocytes.¹²

5 ∣. HOW SIA PROFILE AFFECTS XENOTRANSPLANTATION

Xenotransplantation is viewed as an attractive potential solution to the human organ shortage, which currently limits the availability of organ, cell, and tissue transplantation. Based on their size and breeding characteristics, pigs have been proposed as the most promising donor species for application in man. However, humans generally have high levels of preformed “natural” antibodies that recognize the porcine Galactose-α-1,3-galactose (αGal) antigen as well other carbohydrate antigens expressed by pigs that are not found in humans such as Neu5Gc. Binding of anti-αGal and other anti-pig antibodies to pig cell surfaces initiates complement activation, resulting in hyperacute rejection (HAR) of wild-type porcine organs.^13,14

To overcome this “innate immune” barrier, several modifications have been made to the porcine genome. These include knockout of α-1,3 galactosyltransferase enzyme (GalTKO), removing the αGal epitope from the surface of pig cells; transgenic expression of human complement regulatory proteins such as CD46 (hCD46) and CD55 (hCD55), to inhibit complement injury to cells on which anti-pig antibody is bound; and human endothelial cell protein C receptor (hEPCR), human tissue factor pathway inhibitor (hTFPI), and human thrombomodulin (hTBM), which inhibit non-physiologic activation of clotting mechanisms that are triggered when pig endothelium is exposed to human blood. Each of these modifications improves organ survival in various models. However, alone or even in various combinations, they do not consistently prevent long-term immunologic injury.^15,16

The known mechanisms which contribute to the residual injury include binding of “non-αGal” anti-pig (“xeno-reactive”) antibodies to Sia such as Neu5Gc. The human immune system detects Neu5Gc residues expressed on pig cells as a non-self-carbohydrate antigen (as seen in Figure 1A), perhaps as a defense against translocation of gut bacteria expressing this carbohydrate or tumors expressing Neu5Gc.^17,18 Anti-Neu5Gc antibodies contribute to organ injury during xenotransplantation, as demonstrated by Padler-Karavani and Varki where Neu5Gc deficient mice were induced to make anti-Neu5Gc antibodies, and the induced anti-Neu5Gc antibodies led to the rejection of transplanted allogenic islets expressing Neu5Gc.^19,20 This antigenic target became even more relevant recently as studies have shown that knocking out of α-1,3-galactosyltransferase is associated with increased Neu5Gc levels.²¹ Lutz et al²² recently reported that a significant portion of human IgM and nearly all IgG binding to GalTKO pigs is directed against Neu5Gc and that anti-Neu5Gc antibodies are responsible for significant injury to GalTKO cells and organs. Altogether, experimental evidence so far strongly supports that the recognition of Neu5Gc as a xenoantigen represents an important part in antibody-dependent injury to the pig organ.

FIGURE 1.

Known and predicted roles for sialic acid biology in xenotransplantation. This figure summarizes the known and predicted roles for sialic acid in xenotransplantation. Potential therapeutic approaches that could prevent sialic acid-dependent mechanisms of xenograft injury are illustrated, and include genetic modifications of the pig and pharmacologic interventions. (A) Neu5Gc is present on wild-type and GalTKO pig cells. Like the αGal antigen, Neu5Gc is a carbohydrate that is recognized by preformed “natural” anti-Neu5Gc antibodies, one that contributes to antibody-mediated rejection of pig organ xenografts. Knockout of the gene encoding the enzyme responsible for converting Neu5Ac to Neu5Gc, cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), addresses this problem: GalTKO/CMAHKO pigs do not express Neu5Gc or the αGal antigen. Consequently, these pigs only express Neu5Ac, as do humans; therefore, the human anti-Neu5Gc antibodies have no target to bind to on pig cells.²³ (B) Galectins are galactose-binding lectins expressed by many different cell types; they are also found in soluble form. By binding to desialylated cell surfaces as well as to other receptors, they exert pleiotropic effects, either increasing or decreasing immune responses depending on the cell types in question, their specific galectin expression profile, and the physiologic environment. Our working hypothesis poses that galectins expressed by neutrophils bind to galactose residues exposed by desialylated pig endothelial cells, thereby enhancing the activation and tissue infiltration of human neutrophils into pig tissues. This interaction could be prevented by inhibition of cellular desialylation using NEU inhibitors, or by selective blockade of the galectin-galactose interaction. (C) Asialoglycoprotein receptor-1 (ASGR-1), expressed by Kupffer cells (macrophages), hepatocytes, and liver sinusoidal endothelial cells (LSEC), recognizes galactose on human platelets after Neu5Ac has been cleaved by human or pig sialidases (NEU1-4), causing human platelets to be phagocytosed. This interaction can be prevented by inhibition of platelet desialylation using NEU inhibitors, or by targeting ASGR-1, using either a competitive inhibitor such as asialofetuin, or a blocking antibody (αASGR-1).^53,54 (D) Sia-binding immunoglobulin-type lectins (Siglecs) are a class of Immunoglobulin superfamily receptors binding to sialic acid. In xenotransplantation, pig Siglec-1 (Sialoadhesin, CD169), which is expressed by macrophages, recognizes Neu5Ac on human red blood cells and causes human RBCs to be phagocytosed. RBC phagocytosis is attenuated by blockade of pig sialoadhesin using an anti-CD169 antibody⁶²

On the other hand, Ajit Varki and collaborators recently found that the loss of Neu5Gc primes macrophages toward a more inflammatory and phagocytic state.²³ These findings are potentially relevant to xeno-organs which contain a large proportion of macrophages, such as the lung and liver, and suggest that the role of Neu5Gc in xenotransplantation may extend beyond its role as a xenoantigen. The relevance of this finding needs to be considered in light of the beneficial effects our group and others have observed in Neu5GcKO lung²⁴ and liver²⁵ xeno-perfusion models. We conclude that more research is warranted to improve our understanding of the role of Neu5Gc in xenogeneic conditions and to clarify mechanisms thereby Neu5Gc knockout pigs may limit xenogeneic injury.

Finally, in addition to a role for the specific type of Sia expressed (Neu5Ac vs. Neu5Gc), the overall state of cellular sialylation may be involved in the regulation of inflammatory processes during xenotransplantation such as modulating cell adhesion and trafficking (see below).

6 ∣. MECHANISMS OF SIA-MEDIATED MODULATION OF CELL-CELL INTERACTIONS

Sia reduces cell-to-cell interaction through negative charge-mediated repulsion or “veiling” of adhesion molecules. During xenotransplantation, the Sia profile may become altered by an increase in sialidase activity, which can lead to a pathological increase in cell-to-cell interactions causing or contributing to platelet aggregation, neutrophil/platelet aggregation, and neutrophil adhesion to endothelium.

Platelets contain many pro-inflammatory molecules, and their release can initiate or amplify an inflammatory response. If adhesion of either platelets or neutrophils to endothelium occurs, their aggregates produce an array of molecules known to cause transplant-related tissue damage²⁶ including neutrophil elastase, IL-1β, and reactive oxygen species (ROS). Mandic et al²⁷ believe that lower Sia content on platelets could be directly associated with increased platelet aggregates due to the lower negative surface charge resulting in less repulsion. Platelets steadily lose surface Sia as they age, at least partially through cleavage by sialidases.²⁸ Resting platelets contain an internal pool of sialidase activity which can cause hydrolyzation of terminal Sia from platelet glycoproteins; sialidase activity is dramatically accelerated by platelet activation, and by refrigeration. Platelets express TLR4 which is essential for lipopolysaccharide (LPS)-induced platelet accumulation in the lungs. Feng et al²⁹ found that the two components of the TLR4 complex, TLR4 and MD2, express sialyl residues. Cleavage of these sialyl residues by NEU1 heightens LPS-induced TLR4 complex-initiated signaling which increases platelet accumulation. The translocation of NEU1 to erythrocyte plasma membranes, with the presence of NEU3, is involved in the aggregation of erythrocytes with themselves and endothelial cells.¹²

From this data, we infer that the presence of Sia probably inhibits platelet aggregation during xenotransplantation, while desialylation likely promotes platelet adhesion and activation, and thus thrombus propagation. These interconnected hypotheses are under active investigation by our group.

PMNs express sialidase activity that influences their adhesion to endothelium. Activation of PMNs prompts the translocation of intracellular sialidases to the PMNs surface, which is one of the causes of PMN recruitment in a murine model of sepsis. The mobilization of sialidases to the surface from the lysosome in activated PMNs caused desialylation of the adjacent endothelial surface in vitro and increased resting PMNs adhesion.³⁰ In relation to xenotransplantation, it has been shown that human PMNs bind to naïve porcine endothelium more avidly than to human endothelium under static conditions³¹ through a mechanism that may be CD82-dependent.³²

The role of desialylation in xenogeneic PMNs adhesion is unknown but based on this data it could be playing a major role in the increased PMN adhesion seen during xenotransplantation such as in ex vivo lung perfusions. This is currently a major area of focus in our laboratory.

7 ∣. SELECTINS

Sias in the sequence of Siaα2,3 Galβ1-4(Fucα1-3)GlcNAcβ1-R (sialyl-Lewis(x/a)) are found expressed on leukocytes, platelets, and endothelium. Sialyl-Lewis(x/a) is a component of the ligands for L, P, and E-selectins. Therefore, Sia is necessary for lymphocyte homing, platelet binding and PMN migration.³³ E- and P-selectin expressions are increased on vascular endothelium during inflammation, whereas P-selectin is also expressed on activated platelets, and L-selectin is expressed on activated leukocytes. P-selectin is stored in endothelial cell Weibel-Palade bodies and platelet αgranules and is translocated to the cell surface upon activation. E-selectin has to be transcribed, translated, and transported to the surface. Selectin ligand production requires many factors, importantly including ST.^34-36 In a recent publication, Yang et al³⁷ found that ST ST3Gal-VI contributes to the generation of P-, E-, and L-selectin ligands using ST3Gal-VI-deficient and ST3Gal-IV/ST3Gal-VI double-deficient mice. They also showed that both ST3Gal-VI and ST3Gal-IV provide the majority of the sialylation required for P-selectin ligands and PMNs binding to E-selectin. The overexpression of a ST, ST6GalNAc-II, in a neutrophil-like cell line (HL-60 cells) resulted in reduced rolling on P- and L-selectin under flow conditions.³⁸ Therefore, in this context, Sia is required for cell adhesion, and cellular desialylation would decrease selectin-dependent adhesive interactions.

Which STs are present in specific genetically modified pigs (comparing GalTKO vs. GalTKO.CMAHKO) have not yet been fully investigated. Nor is it known which STs are activated during xenotransplantation³⁹ or how the Sia pathway affects the amount of selectin-mediated and selectin-independent binding of human hematopoietic lineage cells to porcine endothelium. However, based on the data above, this could be an important factor that needs be explored in xenotransplantation as changes in ST could be causing increased selectin-mediated binding of PMNs or other cells types. Alternatively, desialylation may significantly diminish the role of selectins in physiologic regulation of adhesion and cell trafficking, and augment the importance of other pathways such as integrins and galectins.

8 ∣. INTEGRINS

Cellular sialylation also regulates integrin-mediated cellular adhesion on both sides of the receptor/ligand pair. Desialylation of the β2 integrin (CD11b/CD18) exposed an activation epitope on CD18 and increased binding to the integrin ligand Intercellular Adhesion Molecule 1 (ICAM-1), causing increased PMNs binding to ICAM-1. Similarly, desialylation of ICAM-1 greatly enhanced the binding of PMNs under conditions of physiologic shear stress.³³

9 ∣. GALECTINS

One of the central mechanisms involved in intercellular adhesion is mediated by the binding of galectins. Galectins bind to galactose residues, which become exposed on the surface of a wide variety of glycoproteins and glycolipids on cell surfaces after Sia is cleaved. There are twelve known galectins in humans. Galectins bind to many different substrates (such as galactose, integrins, fibronectin, LAMP-1) with varying affinity. Galectins are expressed in a variety of tissues, including endothelial cells, alveolar macrophages, and PMNs and other hematopoietic lineage cells.⁴⁰

Galectins play an important part in PMNs recruitment/activation. When galectin-1 is injected into the peritoneal cavity PMN recruitment is increased, even in the absence of other inflammatory insults. This effect is independent of a G-protein-coupled receptor, and instead has been attributed to the sialoglycoprotein CD43.⁴⁰ In a model of zymosan induced acute inflammation, galectin-1 was expressed and colocalized with L-selectin and β2-integrin both on the plasma membrane and in the cytoplasm of PMNs. Another group found opposite results, in that treatment with galectin-1 inhibited PMN migration and diminished expression of adhesion molecules such as β2-integrin and IL-1β release by peritoneal cells.⁴¹ Galectin-3 has been shown to affect PMN migration and activation, inducing the expression of L-selectin and IL-8 by PMNs in vitro, thereby modifying PMN migration and activation.⁴²

Galectins have also been found to play a role in platelet adhesion. Galectin-8 binds glycans on the platelet membrane and prompts spreading, calcium mobilization and fibrinogen binding, while also promoting platelet aggregation, thromboxane generation, P-selectin expression, and granule secretion. Galectin-1 and −8 promote platelet adhesion by stimulating the transition of αIIbβ3 integrin from a low-affinity/“resting” state to a high-affinity/“active” state, which results in unmasking of epitopes for fibrinogen to bind to.⁴³ Fibrinogen acts as a bridging molecule between platelets to form aggregates. Galectin-1, −3, and −8 all strongly induce P-selectin expression by platelets. Activation of platelets by galectin-1 in the presence of PMNs results in formation of heterotypic cell aggregates in a dose-dependent manner.⁴⁴ Similar mechanisms may be responsible for the increased binding of human PMNs and platelets to xeno-endothelium, further linking sialylation state to the procoagulant phenotype and platelet and PMNs activation events that are associated with xenograft injury. We infer that galectin blockade could prevent a clinically significant part of the inflammatory responses to a xenograft, as illustrated in Figure 1B.

Galectins can also dampen immune responses. Thiemann et al⁴⁵ have found that galectin-1 inhibits tissue migration of immunogenic, but not tolerogenic, dendritic cells. Galectin-3 induces phosphatidylserine exposure on activated T cells causing apoptosis. Galectin-1 does not affect T-cell viability, but it does induce IL-10 production and reduces IFN-ɣ production in activated T cells.⁴⁶ Galectin-8 enables antigen-specific differentiation of Tregs by triggering TGF-β signaling and stimulating sustained IL-2R signaling. Tregs that are differentiated in the presence of galectin-8 express IL-10 at a higher occurrence than normal Tregs.⁴⁷

Given that galectins can play a part in both increasing and decreasing inflammatory and immune responses, understanding their role and regulation in the setting of xenotransplantation is potentially very important. As discussed above, we expect that cellular desialylation could be occurring during a xeno-perfusion or transplantation, leading to the exposure of subjacent galactose residues thereby generating the substrate for galectin binding. Increased amounts of galectins, such as galectin-3 or galectin-8 could be produced during xeno-perfusions or transplantations due to the inflammatory response and could amplify PMN and platelet adhesion by binding to exposed galactose residues expressed by desialylated cells. Therefore, a potential target to present these limitations is to block galectin binding. Such concepts are currently under study in our group. Alternatively, promoting mechanisms by which galectins diminish the immune response could be used as therapy to control adaptive immune responses in xenotransplantation.

10 ∣. MACROPHAGES AND SIA

Pulmonary intravascular macrophages (PIMs) are a specific type of macrophages in angulated animal species that adhere to the lung endothelium and filter the blood of any foreign material or bacteria. It has been shown that depletion of PIMs during xenotransplantation inhibits thromboxane elaboration, complement activation, and histamine and TNF release leading to an increase in xenograft survival.^48-50This strongly suggests that macrophages play an important role in organ rejection during xenotransplantation.

Sia removal from the cell surface results in exposure of underlying glycans that can then be recognized by lectin receptors such as galactose-binding proteins of macrophages (eg asialoglycoprotein receptor-1 (ASGR-1). ASGR-1, a receptor expressed by Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells (LSEC), facilitates phagocytosis of platelets. ASGR-1 recognizes desialylated platelets and binds them to signal phagocytosis as seen in Figure 1C. Paris et al⁵¹ found that in vitro treatment of primary porcine LSEC with siRNA against ASGR-1 transcripts or blocking ASGR-1 with antibodies decreased the ability of porcine LSEC to both bind to and to phagocytose human platelets. They also discovered that there are differences between human and porcine ASGR-1 and that human platelets have four times more exposed galactose β1-4 N-acetyl glucosamine (Galβ) and N-acetyl glucosamine β1-4 N-acetyl glucosamine (βGlcNAc) than fresh porcine platelets. The presence of sialic acid was suggested to shield galactose and N-acetyl glucosamine oligosaccharides, protecting platelets from uptake by ASGR1 and Mac-1, respectively. Indeed, it was found that pig ASGR1 mediates binding and phagocytosis of human platelets that can be inhibited by asialofetuin or an anti-ASGR-1 antibody.^52,53 Recently, it has been shown that ASGR1 knockout pig livers exhibit decreased human platelet uptake.⁵⁴ In aggregate, this data clearly demonstrate a direct connection between ASGR1 and the human (or baboon) platelet uptake seen during liver xenotransplantation.

11 ∣. SIGLECS

Sia-binding immunoglobulin-type lectins (Siglecs) are cell surface proteins that bind Sia and are found primarily on the surface of immune cells such as macrophages, B cells, neutrophils, and NK cells. Most Siglecs contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytosolic region that acts to downregulate signaling pathways and inhibits immune cell activation. Another category of Siglecs mediates cellular adhesion through the recognition of specific sugar moieties on certain cell types. A member of the latter category of Siglecs, sialoadhesin (Siglec-1, CD169), was found to play a role in xenotransplantation.⁵⁵ Rees et al⁵⁶ discovered the loss of human RBC during extracorporeal xeno-perfusion of porcine livers with human blood, a process the authors qualified as “graft vs. host” immune response. They then demonstrated that macrophage receptors, not antibodies, were responsible for the xenogeneic RBC binding.⁵⁷ Altogether, these seminal studies provided initial evidence that cell surface (and perhaps soluble) lectins play a wider role in xenogeneic immune responses than previously appreciated.⁵⁸ Burlak et al and Brock et al^55,59,60 found that porcine sialoadhesin expressed on Kupffer cells is responsible for binding Neu5Ac on the surface of human RBCs. Sialoadhesin is found on the surface of macrophages and its levels are especially high on macrophages of the lungs, spleen, and liver. Waldman et al⁶¹ demonstrated that the addition of anti-porcine sialoadhesin antibody to a porcine liver xeno-perfusion with human erythrocytes attenuates the loss of human RBCs, as depicted in Figure 1D. These data illustrate that lectin-mediated carbohydrate binding, and particularly Siglec-1, plays an important role in xenotransplantation.^56-58,61

12 ∣. SIA AND COMPLEMENT

Xenografts activate the classical pathway of complement because of xeno-reactive antibodies directed against carbohydrate determinants (αGal, Neu5Gc, B4GalNT) and other determinants causing activation of the classical cascade. Deposition of C3b on the cell surface is a central event in the activation of the complement pathway leading to progression toward the C5 convertase as well as activation of a positive loop through the alternative pathway. Binding of factor C3b to alternative pathway factor B to form the C3 convertase is regulated by complement factor H. It was shown that cell surfaces containing polyanions (such as Sia or heparin) enhance the binding affinity of factor H to C3b, resulting in termination of the alternative pathway. Sias also control the activation of complement on erythrocytes, conferring protection from the spontaneous “tick over” of the alternative pathway.⁶² Because host cells are usually covered with polyanions such as glycosaminoglycans and Sia, the presence of Sia would provide a way to discriminate host cells from alternative pathway-activating foreign cells and prevent activation of the complement cascade by self.^63,64 Although genetically modified pig models have been developed that are designed to inhibit complement activation and associated injury, complement activation may still play a role in xenotransplant rejection. There are low levels of plasma C3a, which are formed through the activity of C3 convertase, in the GalTKO lung transplants.¹⁵ Factor H binds Neu5Ac in α2-3, but not α2-6 or α2-8 Sia.⁶⁵ It has also been observed that Neu5Gc also enables binding of human factor H.⁶⁶ Recent studies reported an additional role for factor H in regulating the classical complement pathway by masking the binding sites for C1q.⁶⁷ It has been postulated that the presence of Sia on the cell surface acts as a marker of cellular health that may be lost/impaired during inflammation and oxidative stress.

13 ∣. CONCLUSION

In summary, Sias appear to play a key role in xenotransplantation, as they do in various other biologic systems. Sias play a central role in regulating inflammation and cell adhesion, which are both important obstacles not yet solved in xenotransplantation. Sia profile affects pathways central to behavior of porcine cell and organ xenografts transplanted into non-human primates or humans. By better understanding the roles of ASGR-1 and Siglec-1 (Sialoadhesin) on macrophages, and by preventing galectin binding, managing Sia-associated charge effects, and eliminating the impact of anti-Neu5Gc antibodies, their deleterious consequences in xenotransplantation may be prevented. More scientific focus and further research on the Sia pathway are likely to yield improved ways to prevent associated pathology, and prove helpful to bring xenotransplantation to clinical application.

Abbreviations:

ASGR-1

asialoglycoprotein receptor-1

CMAH

CMP-Neu5Ac hydroxylase

αGal

Gal-α-1,3-galactose

Gal

galactose

GalNAc

N-acetylgalactosamine

GalTKO

galactosyl transferase knock-out

GlcNAc

N-acetylglucosamine

HAR

hyperacute rejection

hEPCR

human endothelial cell protein C receptor

hTFPI

human tissue factor pathway inhibitor

hTBM

human thrombomodulin

ITIMS

immunoreceptor tyrosine-based inhibitory motifs

LPS

lipopolysaccharide

LSEC

liver sinusoidal endothelial cells

Neu5Ac

N-acetylneuraminic acid

Neu5Gc

N-glycolylneuraminic acid

NEU

neuraminidase (Sialidase)

PIMs

pulmonary intravascular macrophages

PMNs

neutrophils

ROS

reactive oxygen species

Sia

sialic acid

Sialoadhesin

siglec-1/CD169

Siglecs

sialic acid-binding immunoglobulin-type lectins

ST

sialyltransferase