Targeting inhibitory Siglec-3 to suppress IgE-mediated human basophil degranulation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin-3 (Siglec-3 [CD33]) is a major Siglec expressed on human mast cells and basophils; engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs. OBJECTIVE: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex by using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). METHODS: Direct and indirect basophil activation tests (BATs) were used to assess both antigen-specific (peanut) and antigen-nonspecific (polyclonal anti-IgE) stimulation. Whole blood from donors with allergy was used for direct BAT, whereas blood from donors with nonfood allergy was passively sensitized with plasma from donors with peanut allergy in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for 1 hour or overnight and then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. RESULTS: Incubation for 1 hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to N-formylmethionyl-leucyl-phenylalanine, providing evidence that this inhibition is IgE pathway-specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. CONCLUSIONS: Pretreating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L before antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.

Human brain sialoglycan ligand for CD33, a microglial inhibitory Siglec implicated in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by accumulation of misfolded proteins. Genetic studies implicate microglia, brain-resident phagocytic immune cells, in AD pathogenesis. As positive effectors, microglia clear toxic proteins, whereas as negative effectors, they release proinflammatory mediators. An imbalance of these functions contributes to AD progression. Polymorphisms of human CD33, an inhibitory microglial receptor, are linked to AD susceptibility; higher CD33 expression correlates with increased AD risk. CD33, also called Siglec-3, is a member of the sialic acid-binding immunoglobulin-type lectin (Siglec) family of immune regulatory receptors. Siglec-mediated inhibition is initiated by binding to complementary sialoglycan ligands in the tissue environment. Here, we identify a single sialoglycoprotein in human cerebral cortex that binds CD33 as well as Siglec-8, the most abundant Siglec on human microglia. The ligand, which we term receptor protein tyrosine phosphatase zeta (RPTPζ)S3L, is composed of sialylated keratan sulfate chains carried on a minor isoform/glycoform of RPTPζ (phosphacan) and is found in the extracellular milieu of the human brain parenchyma. Brains from human AD donors had twofold higher levels of RPTPζS3L than age-matched control donors, raising the possibility that RPTPζS3L overexpression limits misfolded protein clearance contributing to AD pathology. Mice express the same structure, a sialylated keratan sulfate RPTPζ isoform, that binds mouse Siglec-F and crossreacts with human CD33 and Siglec-8. Brains from mice engineered to lack RPTPζ, the sialyltransferase St3gal4, or the keratan sulfate sulfotransferase Chst1 lacked Siglec binding, establishing the ligand structure. The unique CD33 and Siglec-8 ligand, RPTPζS3L, may contribute to AD progression.

Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

Genome-wide association studies demonstrated that polymorphisms in the CD33/sialic acid-binding immunoglobulin-like lectin 3 gene are associated with late-onset Alzheimer's disease (AD). CD33 is expressed on myeloid immune cells and mediates inhibitory signaling through protein tyrosine phosphatases, but the exact function of CD33 in microglia is still unknown. Here, we analyzed CD33 knockout human THP1 macrophages and human induced pluripotent stem cell-derived microglia for immunoreceptor tyrosine-based activation motif pathway activation, cytokine transcription, phagocytosis, and phagocytosis-associated oxidative burst. Transcriptome analysis of the macrophage lines showed that knockout of CD33 as well as knockdown of the CD33 signaling-associated protein tyrosine phosphatase, nonreceptor type 6 (PTPN6) led to constitutive activation of inflammation-related pathways. Moreover, deletion of CD33 or expression of Exon 2-deleted CD33 (CD33ΔE2 /CD33m) led to increased phosphorylation of the kinases spleen tyrosine kinase (SYK) and extracellular signal-regulated kinase 1 and 2 (ERK1 and 2). Transcript analysis by quantitative real-time polymerase chain reaction confirmed increased levels of interleukin (IL) 1B, IL8, and IL10 after knockout of CD33 in macrophages and microglia. In addition, upregulation of the gene transcripts of the AD-associated phosphatase INPP5D was observed after knockout of CD33. Functional analysis of macrophages and microglia showed that phagocytosis of aggregated amyloid-ß1-42 and bacterial particles were increased after knockout of CD33 or CD33ΔE2 expression and knockdown of PTPN6. Furthermore, the phagocytic oxidative burst during uptake of amyloid-ß1-42 or bacterial particles was increased after CD33 knockout but not in CD33ΔE2 -expressing microglia. In summary, deletion of CD33 or expression of CD33ΔE2 in human macrophages and microglia resulted in putative beneficial phagocytosis of amyloid ß1-42 , but potentially detrimental oxidative burst and inflammation, which was absent in CD33ΔE2 -expressing microglia.

CD33M inhibits microglial phagocytosis, migration and proliferation, but the Alzheimer's disease-protective variant CD33m stimulates phagocytosis and proliferation, and inhibits adhesion.

CD33 is a Siglec (sialic acid-binding immunoglobulin-type lectin) receptor on microglia. Human CD33 can be alternatively spliced into two isoforms: the long isoform (CD33M) and a shorter isoform (CD33m) that lacks the sialic acid-binding site. CD33m appears to protect against Alzheimer's disease; however, it remains unclear how. To investigate potential mechanisms by which CD33m may confer protection, we expressed the CD33m and CD33M isoforms of human CD33 in mouse BV-2 and human CHME3 microglial cells and assessed microglia functions. In the BV-2 cells, CD33M inhibited microglial phagocytosis of beads, synapses, debris and dead cells, while CD33m increased phagocytosis of beads, debris and cells. RNAi knockdown of the endogenous mouse CD33 increased phagocytosis and prevented CD33m's (but not CD33M's) effect on phagocytosis. CD33M increased cell attachment but inhibited cell proliferation, while CD33m did the opposite. We also found that CD33M inhibited cell migration. In human CHME3 cells, CD33M increased cell attachment, but inhibited phagocytosis, proliferation and migration, whereas CD33m did the opposite. We conclude that CD33M inhibits microglial phagocytosis, inhibits migration and increases adhesion, while CD33m increases phagocytosis, proliferation and inhibits adhesion. Thus, CD33m might protect against Alzheimer's disease by increasing microglial proliferation, movement and phagocytosis of debris and dead cells.

3'-sialyllactose targets cell surface protein, SIGLEC-3, and induces megakaryocyte differentiation and apoptosis by lipid raft-dependent endocytosis.

3'-sialyllactose is one of the abundant components in human milk oligosaccharides (HMOs) that protect infants from various viral infections in early stages of immune system development. 3SL is a combination of lactose and sialic acid. Most sialic acids are widely expressed in animal cells and they bind to siglec proteins. In this study, we demonstrate that 3SL specifically binds to CD33. It induces megakaryocyte differentiation and subsequent apoptosis by targeting cell surface protein siglec-3 (CD33) in human chronic myeloid leukemia K562 cells. The 3SL-bound CD33 was internalized to the cytosol via caveolae-dependent endocytosis. At the molecular level, 3SL-bound CD33 recruits the suppressor of cytokine signaling 3 (SOCS3) and SH2 domain-containing protein tyrosine phosphatase 1 (SHP1). SOCS3 is degraded with CD33 by proteasome degradation, while SHP-1 activates extracellular signal-regulated kinase (ERK) to induce megakaryocytic differentiation and subsequent apoptosis. The present study, therefore, suggests that 3SL is a potential anti-leukemia agent affecting differentiation and apoptosis.

The Alzheimer's disease-protective CD33 splice variant mediates adaptive loss of function via diversion to an intracellular pool.

The immunomodulatory receptor Siglec-3/CD33 influences risk for late-onset Alzheimer's disease (LOAD), an apparently human-specific post-reproductive disease. CD33 generates two splice variants: a full-length CD33M transcript produced primarily by the "LOAD-risk" allele and a shorter CD33m isoform lacking the sialic acid-binding domain produced primarily from the "LOAD-protective" allele. An SNP that modulates CD33 splicing to favor CD33m is associated with enhanced microglial activity. Individuals expressing more protective isoform accumulate less brain ß-amyloid and have a lower LOAD risk. How the CD33m isoform increases ß-amyloid clearance remains unknown. We report that the protection by the CD33m isoform may not be conferred by what it does but, rather, from what it cannot do. Analysis of blood neutrophils and monocytes and a microglial cell line revealed that unlike CD33M, the CD33m isoform does not localize to cell surfaces; instead, it accumulates in peroxisomes. Cell stimulation and activation did not mobilize CD33m to the surface. Thus, the CD33m isoform may neither interact directly with amyloid plaques nor engage in cell-surface signaling. Rather, production and localization of CD33m in peroxisomes is a way of diminishing the amount of CD33M and enhancing ß-amyloid clearance. We confirmed intracellular localization by generating a CD33m-specific monoclonal antibody. Of note, CD33 is the only Siglec with a peroxisome-targeting sequence, and this motif emerged by convergent evolution in toothed whales, the only other mammals with a prolonged post-reproductive lifespan. The CD33 allele that protects post-reproductive individuals from LOAD may have evolved by adaptive loss-of-function, an example of the less-is-more hypothesis.

Plasma proteomic profiling identifies CD33 as a marker of HIV control in natural infection and after therapeutic vaccination.

BACKGROUND: Biomarkers predicting the outcome of HIV-1 virus control in natural infection and after therapeutic interventions in HIV-1 cure trials remain poorly defined. The BCN02 trial (NCT02616874), combined a T-cell vaccine with romidepsin (RMD), a cancer-drug that was used to promote HIV-1 latency reversal and which has also been shown to have beneficial effects on neurofunction. We conducted longitudinal plasma proteomics analyses in trial participants to define biomarkers associated with virus control during monitored antiretroviral pause (MAP) and to identify novel therapeutic targets that can improve future cure strategies. METHODS: BCN02 was a phase I, open-label, single-arm clinical trial in early-treated, HIV infected individuals. Longitudinal plasma proteomes were analyzed in 11 BCN02 participants, including 8 participants that showed a rapid HIV-1 plasma rebound during a monitored antiretroviral pause (MAP-NC, 'non-controllers') and 3 that remained off ART with sustained plasma viremia <2000 copies/ml (MAP-C, 'controllers'). Inflammatory and neurological proteomes in plasma were evaluated and integration data analysis (viral and neurocognitive parameters) was performed. Validation studies were conducted in a cohort of untreated HIV-1+ individuals (n = 96) and in vitro viral replication assays using an anti-CD33 antibody were used for functional validation. FINDINGS: Inflammatory plasma proteomes in BCN02 participants showed marked longitudinal alterations. Strong proteome differences were also observed between MAP-C and MAP-NC, including in baseline timepoints. CD33/Siglec-3 was the unique plasma marker with the ability to discriminate between MAPC-C and MAP-NC at all study timepoints and showed positive correlations with viral parameters. Analyses in an untreated cohort of PLWH confirmed the positive correlation between viral parameters and CD33 plasma levels, as well as PBMC gene expression. Finally, adding an anti-CD33 antibody to in vitro virus cultures significantly reduced HIV-1 replication and proviral levels in T cells and macrophages. INTERPRETATION: This study indicates that CD33/Siglec-3 may serve as a predictor of HIV-1 control and as potential therapeutic tool to improve future cure strategies. FUNDING: Spanish Science and Innovation Ministry (SAF2017-89726-R and PID2020-119710RB-I00), NIH (P01-AI131568), European Commission (GA101057548) and a Grifols research agreement.

A CD33 frameshift variant is associated with neuromyelitis optica spectrum disorders.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is a rare neuroimmunology disorder predominantly affecting the East Asia population, the reason for this preference remains unknown. Genetic factors such as polymorphisms in human leukocyte antigen (HLA) and interleukins (IL) genes have been reported. Although the familial occurrence of NMOSD is rare, it supports that genetic factors may play a role. METHODS: Whole exome sequencing (WES) study was performed on the affected mother and daughter, as well as the unaffected father in a Taiwanese family with NMOSD. A cohort of 19 sporadic patients with aquaporin 4 antibody (AQP4-Ab) positive NMOSD was also recruited; all fulfilled the 2015 International NMOSD Diagnosis Criteria. Sanger sequencing was performed on exon 4 of the CD33 gene on the sporadic NMOSD cohort. RESULTS: WES study revealed a 19 base pair deletion in exon 4 of the CD33 gene, resulting in frameshift premature truncating protein, which segregated with the affected status. CD33 was the most likely candidate gene due to its known function in immune regulation. A total of 19 sporadic NMOSD patients were tested using Sanger sequencing, including 3 patients with other concomitant autoimmune disorders. Two additional NMOSD patients were found to have the same CD33 frameshift variant, which accounts for 19.04% of all NMOSD patients, and 15% following correction for the familial cases; compared to 2% in Taiwanese population controls. CONCLUSION: In this study, we identified a 19 base pair deletion in the CD33 gene may be a potential risk locus for NMOSD, which is predicted to cause loss of function of CD33. The loss of CD33 inhibitory function may affect the regulation of the immune system in NMOSD patients. This finding requires further larger cohorts of NMOSD patients and functional study to corroborate.

Molecular and functional characterization of porcine Siglec-3/CD33 and analysis of its expression in blood and tissues.

A cDNA clone encoding a 380 a-a type 1 transmembrane protein with homology to human Siglec-3/CD33 was obtained from a swine small intestine library. An analysis of protein sequence identified two immunoglobulin-like domains, a transmembrane region, and a carboxi-terminal tail with two tyrosine-based signalling motifs. Binding assays of Siglec-3 transfected CHO cells to polyacrylamide glycoconjugates showed a preference for α2-6-linked sialic acids. Using mAbs raised against a fragment containing the two Ig-like domains, porcine Siglec-3 was found to be expressed on monocytes and granulocytes, and their bone marrow precursors. It was also detected in lymph node, splenic and alveolar macrophages. MAbs immunoprecipitated, from granulocyte lysates, a protein of 51-60 kDa under both non-reducing and reducing conditions. MAbs were also used to analyse functional activity of Siglec-3 on bone marrow and blood cells. Engagement of Siglec-3 by mAb had no apparent effect on cell proliferation or cytokine production.

Association of CD33 polymorphism rs3865444 with Alzheimer's disease pathology and CD33 expression in human cerebral cortex.

Recent findings identified the minor A allele present in the single-nucleotide polymorphism rs3865444 in the CD33 gene as being associated with the reduced risk of developing Alzheimer's disease (AD). CD33 (Siglec-3) is an immune function protein with anti-inflammatory signaling, cell adhesion, and endocytosis functions with sialic acid-modified proteins or lipids as ligands. Its involvement in AD pathologic mechanisms is still unclear; so, the goal of this study was to investigate if the rs3865444 polymorphism affects the development of AD pathology and the expression of CD33 messenger RNA (mRNA) and protein. For this study, we used DNA from 96 nondemented (ND) and 97 AD neuropathologically diagnosed cases to identify the different rs3865444 alleles and correlate with different measures of AD pathology. Using semiquantitative histologic measures of plaque and tangle pathology, we saw no significant differences between the different genotypes within these disease groups. However, increased expression of CD33 mRNA was associated with increasing AD pathology in temporal cortex brain samples. We also showed that cases with A/A alleles had reduced levels of CD33 protein in temporal cortex but increased levels of the microglia protein IBA-1. Using immunohistochemistry on temporal cortex sections, CD33 was selectively localized to microglia, with greater expression in activated microglia. The factors causing increased CD33 expression by microglia in brain are still unclear, although both genetic and disease factors are involved. Treatment of human microglia isolated from autopsy brains with amyloid-beta peptide and a range of other inflammatory activating agents resulted in reduced CD33 mRNA and protein levels.