Self-associated Molecular Patterns Research Articles

"Osteo-Organogenesis Niche" Hyaluronic Acid Engineered Materials Directing Re-Osteo-Organogenesis via Manipulating Macrophage CD44-MAPK/ERK-ETV1/5-MRC1 Axis.

The strategy of re-organogenesis provides an optimal framework for restoring complex organ structures and functions in adult damage. While the focus has often been on restoring organogenesis stem cells, there is limited investigations of reverting the environmental niche to support this approach. The guiding principle of "Nature selects the fittest to survive" drives the intricate dynamic changes in cellular events within the niche environment, especially through immune surveillance. The extracellular matrix (ECM) serves as the "self-associated molecular patterns" of the niche, containing extensive data on cell-niche reaction data and acting as the active tuner of immune surveillance. In this study, hyaluronic acid (HA) is identified as a unique component of the ECM in cranial osteo-organogenesis. Mechanistically, HA activates the Cluster of Differentiation 44 (CD44)-Mitogen-Activated Protein Kinase (MAPK)/Extracellular Signal-Regulated Kinase (ERK)-Ets Variant 1/5 (ETV1/5)- Mannose Receptor C-Type 1 (MRC1) axis in macrophages, establishing a distinct immune surveillance during osteo-organogenesis. Furthermore, HA is utilized as a novel engineered material for an "Osteo-organogenesis niche", restoring immune surveillance and synergistically regulating stem cells to achieve re-osteo-organogenesis in cranial defects of rats. Taken together, the study unveils a previously unknown strategy for leveraging re-organogenesis by utilizing "organogenesis niche" ECM engineered materials to manipulate immune surveillance, thereby comprehensively regulating stem cells and other tissue cells effectively for re-organogenesis.

Immune regulatory networks coordinated by glycans and glycan-binding proteins in autoimmunity and infection.

The immune system is coordinated by an intricate network of stimulatory and inhibitory circuits that regulate host responses against endogenous and exogenous insults. Disruption of these safeguard and homeostatic mechanisms can lead to unpredictable inflammatory and autoimmune responses, whereas deficiency of immune stimulatory pathways may orchestrate immunosuppressive programs that contribute to perpetuate chronic infections, but also influence cancer development and progression. Glycans have emerged as essential components of homeostatic circuits, acting as fine-tuners of immunological responses and potential molecular targets for manipulation of immune tolerance and activation in a wide range of pathologic settings. Cell surface glycans, present in cells, tissues and the extracellular matrix, have been proposed to serve as "self-associated molecular patterns" that store structurally relevant biological data. The responsibility of deciphering this information relies on different families of glycan-binding proteins (including galectins, siglecs and C-type lectins) which, upon recognition of specific carbohydrate structures, can recalibrate the magnitude, nature and fate of immune responses. This process is tightly regulated by the diversity of glycan structures and the establishment of multivalent interactions on cell surface receptors and the extracellular matrix. Here we review the spatiotemporal regulation of selected glycan-modifying processes including mannosylation, complex N-glycan branching, core 2 O-glycan elongation, LacNAc extension, as well as terminal sialylation and fucosylation. Moreover, we illustrate examples that highlight the contribution of these processes to the control of immune responses and their integration with canonical tolerogenic pathways. Finally, we discuss the power of glycans and glycan-binding proteins as a source of immunomodulatory signals that could be leveraged for the treatment of autoimmune inflammation and chronic infection.

Evolution of Human-Specific Alleles Protecting Cognitive Function of Grandmothers.

The myelomonocytic receptor CD33 (Siglec-3) inhibits innate immune reactivity by extracellular V-set domain recognition of sialic acid (Sia)-containing “self-associated molecular patterns” (SAMPs). We earlier showed that V-set domain-deficient CD33-variant allele, protective against late-onset Alzheimer’s Disease (LOAD), is derived and specific to the hominin lineage. We now report multiple hominin-specific CD33 V-set domain mutations. Due to hominin-specific, fixed loss-of-function mutation in the CMAH gene, humans lack N-glycolylneuraminic acid (Neu5Gc), the preferred Sia-ligand of ancestral CD33. Mutational analysis and molecular dynamics (MD)-simulations indicate that fixed change in amino acid 21 of hominin V-set domain and conformational changes related to His45 corrected for Neu5Gc-loss by switching to N-acetylneuraminic acid (Neu5Ac)-recognition. We show that human-specific pathogens Neisseria gonorrhoeae and Group B Streptococcus selectively bind human CD33 (huCD33) as part of immune-evasive molecular mimicry of host SAMPs and that this binding is significantly impacted by amino acid 21 modification. In addition to LOAD-protective CD33 alleles, humans harbor derived, population-universal, cognition-protective variants at several other loci. Interestingly, 11 of 13 SNPs in these human genes (including CD33) are not shared by genomes of archaic hominins: Neanderthals and Denisovans. We present a plausible evolutionary scenario to compile, correlate, and comprehend existing knowledge about huCD33-evolution and suggest that grandmothering emerged in humans.

Glycans as a key factor in self and nonself discrimination: impact on the breach of immune tolerance.

Glycans are carbohydrates that are made by all organisms and covalently conjugated to other biomolecules. Glycans cover the surface of both human cells and pathogens and are fundamental to defining the identity of a cell or an organism, thereby contributing to discriminating self from nonself. As such, glycans are a class of 'Self-Associated Molecular Patterns' that can fine-tune host inflammatory processes. In fact, glycans can be sensed and recognized by a variety of glycan-binding proteins (GBP) expressed by immune cells, such as galectins, siglecs, and C-type lectins, which recognize changes in the cellular glycosylation, instructing both pro-inflammatory and anti-inflammatory responses. In this review, we introduce glycans as cell-identification structures, discussing how glycans modulate host-pathogen interactions and how they can fine-tune inflammatory processes associated with infection, inflammation and autoimmunity. Finally, from the clinical standpoint, we discuss how glycoscience research can benefit life sciences and clinical medicine by providing a source of valuable biomarkers and therapeutic targets for immunity.

Elucidating Carbohydrate-Protein Interactions Using Nanoparticle-Based Approaches.

Carbohydrates are present on every living cell and coordinate important processes such as self/non-self discrimination. They are amongst the first molecular determinants to be encountered when cellular interactions are initiated. In particular, they resemble essential molecular fingerprints such as pathogen-, danger-, and self-associated molecular patterns guiding key decision-making in cellular immunology. Therefore, a deeper understanding of how cellular receptors of the immune system recognize incoming particles, based on their carbohydrate signature and how this information is translated into a biological response, will enable us to surgically manipulate them and holds promise for novel therapies. One approach to elucidate these early recognition events of carbohydrate interactions at cellular surfaces is the use of nanoparticles coated with defined carbohydrate structures. These particles are captured by carbohydrate receptors and initiate a cellular cytokine response. In the case of endocytic receptors, the capturing enables the engulfment of exogenous particles. Thereafter, the particles are sorted and degraded during their passage in the endolysosomal pathway. Overall, these processes are dependent on the nature of the endocytic carbohydrate receptors and consequently reflect upon the carbohydrate patterns on the exogenous particle surface. This interplay is still an under-studied subject. In this review, we summarize the application of nanoparticles as a promising tool to monitor complex carbohydrate-protein interactions in a cellular context and their application in areas of biomedicine.

PAMPs, DAMPs and SAMPs: Host Glycans are Self‐Associated Molecular Patterns, but subject to Microbial Molecular Mimicry

It has long been known that Pathogen-Associated Molecular Patterns (PAMPs, e.g., lipo-oligosaccharides, LOS) and Danger-Associated Molecular Patterns (DAMPs, e.g., fragments of endogenous hyaluronan) can be glycans or modified glycans. We have more recently emphasized that host glycans can also serve as Self-Associated Molecular Patterns (SAMPs), e.g., sialic acid-bearing glycans that are recognized by CD33-related Siglecs on innate immune cells to dampen responses, or by complement Factor H to dampen complement-mediated activation. However, several commensal or pathogenic organisms have evolved successful molecular mimicry of sialylated SAMPs, so as to hijack these mechanisms and suppress innate immune responses against them. Paired activating CD33rSiglecs have emerged in some lineages, presumably to counter hijacking by microbial molecular mimicry of sialylated SAMPs. Meanwhile, sialylated host SAMPs also have to evolve rapidly to escape from other pathogens that utilize them as targets to initiate infection. The ongoing pressures of these often-competing selection forces can explain our finding that the sialic-acid binding domains of CD33rSiglecs and Factor H show much higher than expected Ka/Ks ratios (an excess of non-synonymous substitutions changing encoded amino acids, over synonymous substitutions). Examples can also be found of lineage-specific complete loss of sialic binding e.g., via mutation of an essential arginine residue in some CD33rSiglec binding pocket––or even whole-sale elimination of the binding domain e.g., alternate splicing out of the amino-terminal V-set domain of CD33/Siglec-3. Such evolutionary changes can be either fixed or polymorphic within a given species. Further complexity arises because CD33rSiglec and Factor H can be directly engaged via protein:protein interactions, both with endogenous proteins such as Hsp70 and Vimentin, and with surface proteins of commensals e.g., the PorB protein of N. gonorrhea. The overall outcome is inter-species or intra-species variations in the intensity of innate immune responses, which can be reflected in relative susceptibility or resistance to infectious agents and/or harmful aggravation of endogenous inflammatory diseases. Perhaps because of the major changes in the sialome resulting from inactivation of the CMAH gene in the Homo lineage leading to humans, these and many other types of evolutionary changes appear to be very prominent in our species and in our pathogen regimes. This presentation will provide an overview of these topics and examples of our recent work in these areas. Varki, A.: Since there are PAMPs and DAMPs, there must be SAMPs? Glycan “self-associated molecular patterns” dampen innate immunity, but pathogens can mimic them. Glycobiology, 21:1121-1124, 2011. Padler-Karavani, V., Hurtado-Ziola, N., Chang, Y.-C., Sonnenburg, J., Ronaghy, A., Yu, H., Verhagen, A., Nizet, V., Chen, X., Varki, N., Varki, A., Angata, T.: Rapid Evolution of Binding Specificities and Expression Patterns of Inhibitory CD33-related Siglecs in Primates. FASEB J. 28:1280-93, 2014. Laubli, H. and Varki, A. Sialic acid–binding immunoglobulin-like lectins (Siglecs) detect self-associated molecular patterns to regulate immune responses. Cell. Mol. Life Sci. Springer, International. 2019.

α2-3 Sialic acid binding and uptake by human monocyte-derived dendritic cells alters metabolism and cytokine release and initiates tolerizing T cell programming.

SummaryDendritic cells (DCs) are key in the initiation of the adaptive T cell responses to tailor adequate immunity that corresponds to the type of pathogen encountered. Oppositely, DCs control the resolution phase of inflammation and are able to induce tolerance after receiving anti-inflammatory cytokines or upon encounter of self-associated molecular patterns, such as α2-3 linked sialic acid (α2-3sia).Objective: We here investigated whether α2-3sia, that bind immune inhibitory Siglec receptors, would alter signaling and reprogramming of LPS-stimulated human monocyte-derived DCs (moDCs).Methods and Results: Transcriptomic analysis of moDCs stimulated with α2-3sia-conjugated dendrimers revealed differentially expressed genes related to metabolic pathways, cytokines, and T cell differentiation. An increase in genes involved in ATPase regulator activity, oxidoreductase activity, and glycogen metabolic processes was detected. Metabolic extracellular flux analysis confirmed a more energetic moDC phenotype upon α2-3sia binding as evidenced by an increase in both glycolysis and mitochondrial oxidative phosphorylation. TH1 differentiation promoting genes IFNL and IL27, were significantly downregulated in the presence of α2-3sia. Functional assays confirmed that α2-3sia binding to moDCs induced phosphorylation of Siglec-9, reduced production of inflammatory cytokines IL-12 and IL-6, and increased IL-10. Surprisingly, α2-3sia-differentiated moDCs promoted FoxP3+CD25+/-CD127- regulatory T cell differentiation and decreased FoxP3-CD25-CD127- effector T cell proliferation.Conclusions: In conclusion, we demonstrate that α2-3sia binding to moDCs, phosphorylates Siglec-9, alters metabolic pathways, cytokine signaling, and T cell differentiation processes in moDCs and promotes regulatory T cells. The sialic acid-Siglec axis on DCs is therefore, a novel target to induce tolerance and to explore for immunotherapeutic interventions aimed to restore inflammatory processes.

A versatile soluble siglec scaffold for sensitive and quantitative detection of glycan ligands

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. The connections between Siglecs and human disease motivate improved methods to detect Siglec ligands. Here, we describe a new versatile set of Siglec-Fc proteins for glycan ligand detection. Enhanced sensitivity and selectivity are enabled through multimerization and avoiding Fc receptors, respectively. Using these Siglec-Fc proteins, Siglec ligands are systematically profiled on healthy and cancerous cells and tissues, revealing many unique patterns. Additional features enable the production of small, homogenous Siglec fragments and development of a quantitative ligand-binding mass spectrometry assay. Using this assay, the ligand specificities of several Siglecs are clarified. For CD33 (Siglec-3), we demonstrate that it recognizes both α2-3 and α2-6 sialosides in solution and on cells, which has implications for its link to Alzheimer’s disease susceptibility. These soluble Siglecs reveal the abundance of their glycan ligands on host cells as self-associated molecular patterns.

Human-specific microglial Siglec-11 transcript variant has the potential to affect polysialic acid-mediated brain functions at a distance.

CD33-related Siglecs are often found on innate immune cells and modulate their reactivity by recognition of sialic acid-based "self-associated molecular patterns" and signaling via intracellular tyrosine-based cytosolic motifs. Previous studies have shown that Siglec-11 specifically binds to the brain-enriched polysialic acid (polySia/PSA) and that its microglial expression in the brain is unique to humans. Furthermore, human microglial Siglec-11 exists as an alternate splice form missing the exon encoding the last (fifth) Ig-like C2-set domain of the extracellular portion of the protein, but little is known about the functional consequences of this variation. Here, we report that the recombinant soluble human microglial form of Siglec-11 (hSiglec-11(4D)-Fc) binds endogenous and immobilized polySia better than the tissue macrophage form (hSiglec-11(5D)-Fc) or the chimpanzee form (cSiglec-11(5D)-Fc). The Siglec-11 protein is also prone to aggregation, potentially influencing its ligand-binding ability. Additionally, Siglec-11 protein can be secreted in both intact and proteolytically cleaved forms. The microglial splice variant has reduced proteolytic release and enhanced incorporation into exosomes, a process that appears to be regulated by palmitoylation of cysteines in the cytosolic tail. Taken together, these data demonstrate that human brain specific microglial hSiglec-11(4D) has different molecular properties and can be released on exosomes and/or as proteolytic products, with the potential to affect polySia-mediated brain functions at a distance.

Multiple Genomic Events Altering Hominin SIGLEC Biology and Innate Immunity Predated the Common Ancestor of Humans and Archaic Hominins.

Human-specific pseudogenization of the CMAH gene eliminated the mammalian sialic acid (Sia) Neu5Gc (generating an excess of its precursor Neu5Ac), thus changing ubiquitous cell surface “self-associated molecular patterns” that modulate innate immunity via engagement of CD33-related-Siglec receptors. The Alu-fusion-mediated loss-of-function of CMAH fixed ∼2–3 Ma, possibly contributing to the origins of the genus Homo. The mutation likely altered human self-associated molecular patterns, triggering multiple events, including emergence of human-adapted pathogens with strong preference for Neu5Ac recognition and/or presenting Neu5Ac-containing molecular mimics of human glycans, which can suppress immune responses via CD33-related-Siglec engagement. Human-specific alterations reported in some gene-encoding Sia-sensing proteins suggested a “hotspot” in hominin evolution. The availability of more hominid genomes including those of two extinct hominins now allows full reanalysis and evolutionary timing. Functional changes occur in 8/13 members of the human genomic cluster encoding CD33-related Siglecs, all predating the human common ancestor. Comparisons with great ape genomes indicate that these changes are unique to hominins. We found no evidence for strong selection after the Human–Neanderthal/Denisovan common ancestor, and these extinct hominin genomes include almost all major changes found in humans, indicating that these changes in hominin sialobiology predate the Neanderthal–human divergence ∼0.6 Ma. Multiple changes in this genomic cluster may also explain human-specific expression of CD33rSiglecs in unexpected locations such as amnion, placental trophoblast, pancreatic islets, ovarian fibroblasts, microglia, Natural Killer(NK) cells, and epithelia. Taken together, our data suggest that innate immune interactions with pathogens markedly altered hominin Siglec biology between 0.6 and 2 Ma, potentially affecting human evolution.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) detect self-associated molecular patterns to regulate immune responses.

The mammalian immune system evolved to tightly regulate the elimination of pathogenic microbes and neoplastic transformed cells while tolerating our own healthy cells. Here, we summarize experimental evidence for the role of Siglecs-in particular CD33-related Siglecs-as self-receptors and their sialoglycan ligands in regulating this balance between recognition of self and non-self. Sialoglycans are found in the glycocalyx and extracellular fluids and matrices of all mammalian cells and can be considered as self-associated molecular patterns (SAMPs). We also provide an overview of the known interactions of Siglec receptors and sialoglycan-SAMPs. Manipulation of the Siglec-SAMP axis offers new therapeutic opportunities for the treatment of inflammatory conditions, autoimmune diseases and also cancer immunotherapy.

Decreased Erythrocyte Binding Capability for Neutrophil Siglec-9 Is a Source of Oxidative Stress in Sickle Cell Disease

IntroductionOxidative stress and inflammation promote hemolysis and vaso-occlusion in sickle cell disease (SCD). Erythrocytes can play a pro-oxidative and anti-oxidative role in disease-associated inflammation through iron-driven free radical production and endogenous anti-oxidants. In SCD, HbS accelerated auto-oxidation, iron de-compartmentalization, and inflammatory cell-derived oxidants drive this oxidative stress. CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids in “Self-Associated Molecular Patterns” (SAMPs) and typically inhibit innate immune cell functions via cytosolic signaling. Recent studies have shown that Siglec-9 on human neutrophils in circulating blood interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactivity, including reactive oxygen species (ROS) production. Modification of erythrocyte membrane sialic acids interferes with their ability to inhibit neutrophil activation and oxidative burst. As erythrocytes age and undergo cellular damage there is a loss of membrane bound sialoglycoproteins. Several studies have indicated that this reduction in the sialome is accelerated on the sickle erythrocyte. We hypothesize that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and that decreased binding of neutrophil Siglec-9 to sickle erythrocyte sialic acid enhances neutrophil activation and oxidative burst.Methods & ResultsBinding of recombinant Siglec-9-Fc protein to AA (n=9) and SS erythrocytes (n=7) was measured using flow cytometry. SS erythrocytes displayed significantly less Siglec-9-Fc binding 45% ± 11.9 (mean ± SEM) compared to AA erythrocytes 82% ± 5.4 (p=.03). Treatment of AA erythrocytes with neuraminidase to remove sialic acid decreased binding to 4% ± 7.9.Neutrophil ROS production was measured by flow cytometry using dihydrorhodamine 123 (DHR123). Neutrophils were purified from AA donors (n=5) and SS (n=11) or AA erythrocytes (n=5) were added to the neutrophils at a ratio of 50 erythrocytes to 1 neutrophil. Oxidative burst was stimulated using phorbol 12-myristate 13-acetate (PMA). AA erythrocytes decreased PMA-stimulated neutrophil ROS production by 84% ± 6.7. In contrast, SS erythrocytes decreased PMA-stimulated neutrophil ROS production by 53% ± 6.8 (p=0.03,).Recent studies have shown that neutrophil extracellular trap (NET) formation is pathogenic in SCD. We added AA and SS erythrocytes (50:1) to neutrophils stimulated with PMA and NET formation was assessed using Syto Orange, Syto Green and confocal microscopy. PMA-stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation. In contrast, AA erythrocytes treated with neuraminidase to remove sialic acid had increased NET formation. PMA-stimulated neutrophils incubated with SS erythrocytes showed increased NET formation.ConclusionsA constant disease state of oxidative stress and inflammation underlies SCD pathophysiology. These data demonstrate that SS erythrocytes with decreased membrane sialic acid are deficient in binding to neutrophil Siglec-9. The decreased binding of SS erythrocytes to neutrophil Siglec-9 diminishes the ability of SS erythrocytes to properly modulate neutrophil activation, which may contribute to the oxidative stress and increased state of basal inflammation inherent to SCD. The overall reduction in number of RBCs in SS may also be a factor? [Display omitted] DisclosuresBelcher:CSL Behring: Research Funding. Vercellotti:CSL Behring: Research Funding.

Self-associated molecular patterns mediate cancer immune evasion by engaging Siglecs on T cells.

First-generation immune checkpoint inhibitors, including anti-CTLA-4 and anti-programmed death 1 (anti-PD-1) antibodies, have led to major clinical progress, yet resistance frequently leads to treatment failure. Thus, new targets acting on T cells are needed. CD33-related sialic acid-binding immunoglobulin-like lectins (Siglecs) are pattern-recognition immune receptors binding to a range of sialoglycan ligands, which appear to function as self-associated molecular patterns (SAMPs) that suppress autoimmune responses. Siglecs are expressed at very low levels on normal T cells, and these receptors were not until recently considered as interesting targets on T cells for cancer immunotherapy. Here, we show an upregulation of Siglecs, including Siglec-9, on tumor-infiltrating T cells from non-small cell lung cancer (NSCLC), colorectal, and ovarian cancer patients. Siglec-9-expressing T cells coexpressed several inhibitory receptors, including PD-1. Targeting of the sialoglycan-SAMP/Siglec pathway in vitro and in vivo resulted in increased anticancer immunity. T cell expression of Siglec-9 in NSCLC patients correlated with reduced survival, and Siglec-9 polymorphisms showed association with the risk of developing lung and colorectal cancer. Our data identify the sialoglycan-SAMP/Siglec pathway as a potential target for improving T cell activation for immunotherapy.

Erythrocyte Sialoglycoproteins Engage Siglec-9 to Maintain Neutrophil Quiescence in the Bloodstream

Neutrophils remain functionally quiescent in the bloodstream of healthy humans. Characterized by a short lifespan (for which estimates vary widely), they exit the bloodstream to mediate various functions such as pathogen elimination or wound healing--or undergo spontaneous apoptosis and macrophage-mediated clearance in the absence of an inflammatory stimulus. Understanding of neutrophil quiescence, activation and lifespan is relevant to many clinical situations such as infection, sepsis, reperfusion injury, granulocyte transfusions, and other neutrophil-mediated inflammatory pathologies.It has long been known that neutrophils purified from blood tend to be short-lived and show signs of activation, but this technical challenge has been assumed to be a reflection of their natural biology in vivo. We found that when purified from whole blood, neutrophils indeed rapidly express activation markers (low L-selectin; high CD11b) and progress to apoptosis. However, using a novel method to label neutrophils in situ followed by flow cytometry, we found that these activation events occurred at a much slower rate in neutrophils that remained in whole blood. This finding led us to hypothesize that during separation of neutrophils from other blood components, one may be removing an inhibitor that normally maintains neutrophil quiescence in the bloodstream.Co-incubation studies showed that erythrocytes are the primary blood component that dampens neutrophil activation, including chemotaxis, generation of reactive oxygen species, and release of neutrophil extracellular traps. The duration of neutrophil viability was also lengthened upon re-incubation with erythrocytes. We found that this maintenance of functional quiescence is mediated largely by erythrocyte surface sialoglycoproteins (particularly glycophorin A, GPA), which engage neutrophil Siglec-9, a sialic acid-binding receptor that is known to dampen innate immune cell activation via cytosolic inhibitory motifs. Modification of erythrocyte sialic acid side chains using a newly developed gentle method eliminated Siglec-9 binding, and allowed neutrophil activation as measured by reactive oxygen species production.We next sought evidence that this interaction occurs in vivoby studying freshly collected whole blood. Smears made from fresh whole blood showed a high degree of Siglec-9 clustering on neutrophils, which was evident at points of contact with GPA on erythrocytes. This clustering was markedly reduced when smears were made from buffy coat preparations, which involves initial physical separation of neutrophils from erythrocytes during centrifugation.Taken together, this data indicates that erythrocyte sialic acids have an unexpected function as carriers of "self-associated molecular patterns" (SAMPs), regulating innate immunity and maintaining neutrophil quiescence in the bloodstream, apparently by tonic engagement of inhibitory Siglec-9. Notably, this SAMP effect blunts but does not completely inhibit bacterial killing by neutrophils in whole blood, and yet presumably helps to limit unwanted neutrophil inflammatory activation in the bloodstream. Our findings are relevant to many physiological, pathological and clinical situations involving neutrophil biology, and may be useful in reevaluation of prior studies of activation, function and reinfusion-based kinetics that were performed using neutrophils isolated away from whole blood. DisclosuresNo relevant conflicts of interest to declare.

17P - Upregulation of self-associated molecular patterns and Siglec receptors on T cells facilitate cancer immune evasion

17P - Upregulation of self-associated molecular patterns and Siglec receptors on T cells facilitate cancer immune evasion

Autoimmunity--promoting and stabilizing innate immunity 'UNWUCHT'.

Autoimmunity--promoting and stabilizing innate immunity 'UNWUCHT'.

Host and pathogen hyaluronan signal through human siglec-9 to suppress neutrophil activation.

Inhibitory CD33-related Siglec receptors regulate immune cell activation upon engaging ubiquitous sialic acids (Sias) on host cell surface glycans. Through molecular mimicry, Sia-expressing pathogen group B Streptococcus binds inhibitory human Siglec-9 (hSiglec-9) to blunt neutrophil activation and promote bacterial survival. We unexpectedly discovered that hSiglec-9 also specifically binds high molecular weight hyaluronan (HMW-HA), another ubiquitous host glycan, through a region of its terminal Ig-like V-set domain distinct from the Sia-binding site. HMW-HA recognition by hSiglec-9 limited neutrophil extracellular trap (NET) formation, oxidative burst, and apoptosis, defining HMW-HA as a regulator of neutrophil activation. However, the pathogen group A Streptococcus (GAS) expresses a HMW-HA capsule that engages hSiglec-9, blocking NET formation and oxidative burst, thereby promoting bacterial survival. Thus, a single inhibitory lectin receptor detects two distinct glycan "self-associated molecular patterns" to maintain neutrophil homeostasis, and two leading human bacterial pathogens have independently evolved molecular mimicry to exploit this immunoregulatory mechanism. HMW-HA is the first example of a non-sialic acid containing glycan to be recognized by CD33-related Siglecs. HMW-HA engagement of hSiglec-9 attenuates neutrophil activation. Group A Streptococcus exploits hSiglec-9 recognition via its polysaccharide HMW-HA capsule to subvert neutrophil killing.

Rapid evolution of binding specificities and expression patterns of inhibitory CD33‐related Siglecs in primates

Siglecs are sialic acid-binding Ig-like lectins that recognize sialoglycans via amino-terminal V-set domains. CD33-related Siglecs (CD33rSiglecs) on innate immune cells recognize endogenous sialoglycans as "self-associated molecular patterns" (SAMPs), dampening immune responses via cytosolic immunoreceptor tyrosine-based inhibition motifs that recruit tyrosine phosphatases. However, sialic acid-expressing pathogens subvert this mechanism through molecular mimicry. Meanwhile, endogenous host SAMPs must continually evolve to evade other pathogens that exploit sialic acids as invasion targets. We hypothesized that these opposing selection forces have accelerated CD33rSiglec evolution. We address this by comparative analysis of major CD33rSiglec (Siglec-3, Siglec-5, and Siglec-9) orthologs in humans, chimpanzees, and baboons. Recombinant soluble molecules displaying ligand-binding domains show marked quantitative and qualitative interspecies differences in interactions with strains of the sialylated pathogen, group B Streptococcus, and with sialoglycans presented as gangliosides or in the form of sialoglycan microarrays, including variations such as N-glycolyl and O-acetyl groups. Primate Siglecs also show quantitative and qualitative intra- and interspecies variations in expression patterns on leukocytes, both in circulation and in tissues. Taken together our data explain why the CD33rSiglec-encoding gene cluster is undergoing rapid evolution via multiple mechanisms, driven by the need to maintain self-recognition by innate immune cells, while escaping 2 distinct mechanisms of pathogen subversion.

Letter to the Glyco-Forum: Since there are PAMPs and DAMPs, there must be SAMPs? Glycan "self-associated molecular patterns" dampen innate immunity, but pathogens can mimic them

The ∼500-million-year-old adaptive immune system detects foreign (“non-self”) epitopes via B cell-derived antibodies and/or T cell receptor interactions with major histocompatibility complex (MHC)/peptide complexes (Hedrick 2004). Cells of the more ancient innate immune system display receptors that detect foreign glycans, for example, fungal glycan recognition by the macrophage mannose receptor (Stahl and Ezekowitz 1998) or by circulating collectins and pentraxins (Bottazzi et al. 2010). The latter field was revolutionized by definition of “pathogen-associated molecular patterns” (PAMPs; Medzhitov and Janeway 1997), microbial products that can be detected by pattern recognition receptors (PRRs), particularly the Toll-like receptors (TLRs; Beutler 2009), Nod-like receptors (Davis et al. 2011) and dendritic cell receptors such as C-type lectins (Geijtenbeek et al. 2004). Many PAMPs are glycoconjugates (e.g., bacterial lipo-oligosaccharides) or glycan-based polymers (e.g., bacterial peptidoglycans), including bacterial DNA or viral RNA (which are (deoxy)ribose-based polymers). The innate immune system also recognizes “danger-associated molecular patterns” (DAMPs; Matzinger 2002; Chen and Nunez 2010), molecules released during tissue damage, such as heat-shock proteins, high mobility group box 1 (Lotze and Tracey 2005), hyaluronan (HA) fragments (Taylor and Gallo 2006), glycosaminoglycan (GAG)-bearing matrix proteoglycans (Moreth et al. 2010) and certain crystals (Martinon et al. 2009), all of which originate from damaged host cells or matrices. Signals initiated by DAMPs and PAMPs are transduced via similar pathways, activating innate immune inflammatory responses.

Recovery of Adrenal Function after Substitution of Beclomethasone Dipropionate for Oral Corticosteroids

Of 16 steroid-dependent asthmatic patients oral treatment has been discontinued in six and reduced in four after the introduction of beclomethasone dipropionate. Substitution of inhaled beclomethasone for oral steroids was unsuccessful in the remaining six patients. Serial adrenal function studies in the patients whose oral treatment was discontinued showed progressive recovery, and five out of six had a normal response to tetracosactrin stimulation after two months.