Quantitative Proteomics of Polarised Macrophages Derived from Induced Pluripotent Stem Cells.

Macrophages (MΦ) are highly heterogenous and versatile innate immune cells involved in homeostatic and immune responses. Activated MΦ can exist in two extreme phenotypes: pro-inflammatory (M1) MΦ and anti-inflammatory (M2) MΦ. These phenotypes can be recapitulated in vitro by using ligands of toll-like receptors (TLRs) and cytokines such as IFNγ and IL-4. In recent years, human induced pluripotent stem cells (iPSC)-derived MΦ have gained major attention, as they are functionally similar to human monocyte-derived MΦ and are receptive to genome editing. In this study, we polarised iPSC-derived MΦ to M1 or M2 and analysed their proteome and secretome profiles using quantitative proteomics. These comprehensive proteomic data sets provide new insights into functions of polarised MΦ.

High-Yield Human Induced Pluripotent Stem Cell-Derived Monocytes and Macrophages Are Functionally Comparable With Primary Cells.

Macrophages are pivotal effectors of host immunity and regulators of tissue homeostasis. Understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human induced pluripotent stem cell (hiPSC)-derived monocytes and macrophages, as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of macrophage biology and implication in human diseases. In this study, we present a fully optimized differentiation protocol of hiPSC-derived monocytes and granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). We present characterization of iPSC-derived myeloid lineage cells at phenotypic, functional, and transcriptomic levels, in comparison with corresponding subsets of peripheral blood-derived cells. We also highlight the application of hiPSC-derived monocytes and macrophages as a gene-editing platform for functional validation in research and drug screening, and the study also provides a reference for cell therapies.

Siglec and anti-Siglec therapies.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of receptors that bind sialic acids in specific linkages on glycoproteins and glycolipids. Siglecs play roles in immune signalling and exhibit cell-type specific expression and endocytic properties. Recent studies suggest that Siglecs are likely to function as immune checkpoints that regulate responses in cancers and inflammatory diseases. In this review, we discuss strategies to target the Siglec-sialic acid axis in human diseases, particularly cancer, and the possibility of exploiting them for therapeutic intervention.

Siglec-15 recognition of sialoglycans on tumor cell lines can occur independently of sialyl Tn antigen expression.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin expressed on osteoclast progenitors, which plays an important role in osteoclast development and function. It is also expressed by tumor-associated macrophages and by some tumors, where it is thought to contribute to the immunosuppressive microenvironment. It was shown previously that engagement of macrophage-expressed Siglec-15 with tumor cells expressing its ligand, sialyl Tn (sTn), triggered production of TGF-ß. In the present study, we have further investigated the interaction between Siglec-15 and sTn on tumor cells and its functional consequences. Based on binding assays with lung and breast cancer cell lines and glycan-modified cells, we failed to see evidence for recognition of sTn by Siglec-15. However, using a microarray of diverse, structurally defined glycans, we show that Siglec-15 binds with higher avidity to sialylated glycans other than sTn or related antigen sequences. In addition, we were unable to demonstrate enhanced TGF-ß secretion following co-culture of Siglec-15-expressing monocytic cell lines with tumor cells expressing sTn or following Siglec-15 cross-linking with monoclonal antibodies. However, we did observe activation of the SYK/MAPK signaling pathway following antibody cross-linking of Siglec-15 that may modulate the functional activity of macrophages.

Design and characterization of Zweimab and Doppelmab, high affinity dual antagonistic anti-TSLP/IL13 bispecific antibodies.

Patients with severe Th2 type asthma often have a steroid resistant phenotype and are prone to acute exacerbations. Current novel therapies have only marginal therapeutic effects. One of the hypotheses for lack of major efficacy in most patients is targeting only one redundant pathway leaving others active. Hence, we have designed and developed novel highly potent bispecific anti-TSLP/IL13 antibodies called Zweimabs (monovalent bispecific) and Doppelmabs (bivalent bispecific) that concurrently inhibits the signaling by these two cytokines.

Receptor for advanced glycation endproducts (RAGE) deficiency protects against MPTP toxicity.

Parkinson's disease (PD) is a common neurodegenerative disorder of unknown pathogenesis characterized by the loss of nigrostriatal dopaminergic neurons. Oxidative stress, microglial activation and inflammatory responses seem to contribute to the pathogenesis. The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules. The formation of advanced glycation end products (AGEs), the first ligand of RAGE identified, requires a complex series of reactions including nonenzymatic glycation and free radical reactions involving superoxide-radicals and hydrogen peroxide. Binding of RAGE ligands results in activation of nuclear factor-kappaB (NF-κB). We show that RAGE ablation protected nigral dopaminergic neurons against cell death induced by the neurotoxin MPTP that mimics most features of PD. In RAGE-deficient mice the translocation of the NF-κB subunit p65 to the nucleus, in dopaminergic neurons and glial cells was inhibited suggesting that RAGE involves the activation of NF-κB. The mRNA level of S100, one of the ligands of RAGE, was increased after MPTP treatment. The dopaminergic neurons treated with MPP(+) and S100 protein showed increased levels of apoptotic cell death, which was attenuated in RAGE-deficient mice. Our results suggest that activation of RAGE contributes to MPTP/MPP(+)-induced death of dopaminergic neurons that may be mediated by NF-κB activation.

Glycolaldehyde-modified ß-lactoglobulin AGEs are unable to stimulate inflammatory signaling pathways in RAGE-expressing human cell lines.

SCOPE: Advanced glycation endproducts (AGEs) are suspected to stimulate inflammatory signaling pathways in target tissues via activation of the receptor for AGEs. Endotoxins are generally recognized as potential contamination of AGE preparations and stimulate biological actions that are very similar as or identical to those induced by AGEs. METHODS AND RESULTS: In our study, we used glycolaldehyde-modified ß-lactoglobulin preparations as model AGEs and employed two methods to remove endotoxin using either affinity columns or extraction with Triton X-114 (TX-114). Affinity column-purified AGEs retained their ability to stimulate inflammatory signaling as measured by mRNA expression of inflammatory cytokines in the human lung epithelial cell line Beas2b. However, glycolaldehyde-modified AGEs purified by extraction with TX-114 did not show any stimulation of mRNA expression of inflammatory cytokines. The presence of a cell stimulating endotoxin-like activity was demonstrated in the detergent phase after extraction with TX-114, thus indicating that not AGEs but a lipophilic contamination was responsible for the stimulation of inflammatory signaling. CONCLUSION: Our results demonstrate that glycolaldehyde-modified AGEs are unable to induce inflammatory signaling in receptor for AGE-expressing cells. The observed cell-activating activity can be ascribed to an endotoxin-like lipophilic contamination present in AGE preparations and affinity column purification was insufficient to remove this contamination.

Carboxylated N-glycans on RAGE promote S100A12 binding and signaling.

The receptor for advanced glycation end products (RAGE) is a signaling receptor protein of the immunoglobulin superfamily implicated in multiple pathologies. It binds a diverse repertoire of ligands, but the structural basis for the interaction of different ligands is not well understood. We earlier showed that carboxylated glycans on the V-domain of RAGE promote the binding of HMGB1 and S100A8/A9. Here we study the role of these glycans on the binding and intracellular signaling mediated by another RAGE ligand, S100A12. S100A12 binds carboxylated glycans, and a subpopulation of RAGE enriched for carboxylated glycans shows more than 10-fold higher binding potential for S100A12 than total RAGE. When expressed in mammalian cells, RAGE is modified by complex glycans predominantly at the first glycosylation site (N25IT) that retains S100A12 binding. Glycosylation of RAGE and maximum binding sites for S100A12 on RAGE are also cell type dependent. Carboxylated glycan-enriched population of RAGE forms higher order multimeric complexes with S100A12, and this ability to multimerize is reduced upon deglycosylation or by using non-glycosylated sRAGE expressed in E. coli. mAbGB3.1, an antibody against carboxylated glycans, blocks S100A12-mediated NF-kappaB signaling in HeLa cells expressing full-length RAGE. These results demonstrate that carboxylated N-glycans on RAGE enhance binding potential and promote receptor clustering and subsequent signaling events following oligomeric S100A12 binding.

Structural and functional insights into RAGE activation by multimeric S100B.

Nervous system development and plasticity require regulation of cell proliferation, survival, neurite outgrowth and synapse formation by specific extracellular factors. The EF-hand protein S100B is highly expressed in human brain. In the extracellular space, it promotes neurite extension and neuron survival via the receptor RAGE (receptor for advanced glycation end products). The X-ray structure of human Ca(2+)-loaded S100B was determined at 1.9 A resolution. The structure revealed an octameric architecture of four homodimeric units arranged as two tetramers in a tight array. The presence of multimeric forms in human brain extracts was confirmed by size-exclusion experiments. Recombinant tetrameric, hexameric and octameric S100B were purified from Escherichia coli and characterised. Binding studies show that tetrameric S100B binds RAGE with higher affinity than dimeric S100B. Analytical ultracentrifugation studies imply that S100B tetramer binds two RAGE molecules via the V-domain. In line with these experiments, S100B tetramer caused stronger activation of cell growth than S100B dimer and promoted cell survival. The structural and the binding data suggest that tetrameric S100B triggers RAGE activation by receptor dimerisation.

Soluble RAGE blocks scavenger receptor CD36-mediated uptake of hypochlorite-modified low-density lipoprotein.

Engagement of the receptor for advanced glycation end products (RAGE) by its signal transduction ligands evokes inflammatory cell infiltration and activation of the vessel wall. However, soluble RAGE (sRAGE), the truncated form spanning the extracellular binding domain of RAGE, has potent anti-inflammatory properties by acting as a decoy for RAGE ligands. We now show that sRAGE binds with high affinity to atherogenic low-density lipoprotein (LDL) modified by hypochlorous acid (HOCl), the major oxidant generated by the myeloperoxidase-H2O2-chloride system of phagocytes activated during inflammation. We further demonstrate that sRAGE can be coprecipitated with HOCl-LDL from spiked serum. To determine the functional significance of sRAGE binding to HOCl-LDL, cell association studies with macrophages were performed. sRAGE effectively inhibited cellular uptake of HOCl-LDL and subsequent lipid accumulation. Using Chinese hamster ovary cells overexpressing class B scavenger receptor CD36 or SR-BI, two preferential scavenger receptors for HOCl-LDL, we demonstrate that sRAGE only interferes with CD36-mediated uptake of HOCl-LDL. The present findings indicate that sRAGE acts as a sink for HOCl-LDL, which is abundantly present in human atherosclerotic lesions. We propose that sRAGE represents a physiological antagonist that interferes with scavenger receptor-mediated cholesterol accumulation and foam cell formation of macrophages.

Targeting of tumor cells expressing the prostate stem cell antigen (PSCA) using genetically engineered T-cells.

BACKGROUND: Curative therapeutic options for minimal residual disease or advanced tumor stages in prostate cancer (PCa) are still missing. Adoptive transfer of cytotoxic T-cells that have been polyclonally rendered tumor-specific by genetic engineering appears to be a promising immunotherapeutic strategy. Among the numerous prostate tissue/tumor antigens identified during the last years, the "prostate stem cell antigen" (PSCA) is an attractive immunotherapeutic target. It is broadly expressed on the surface of primary PCa cells as well as on PCa metastases. METHODS: To generate a chimeric T-cell receptor (TCR) recognizing PSCA, a monoclonal anti-PSCA antibody was raised and a single-chain fragment (scFv) was prepared. The resulting anti-PSCA scFv 7F5 was fused to the beta2 constant region derived from the beta-chain of a TCR and to the CD3zeta-signaling domain. RESULTS: The chimeric alpha-PSCA-beta2/CD3zeta-TCR, expressed in Jurkat cells, was phosphorylated in the ITAMs of the CD3-zeta chain upon cross-linking by insolublized PSCA. When transduced into a mouse cytotoxic T-cell line, the chimeric receptor specifically activated cytotoxicity against PSCA-positive tumor cells. CONCLUSIONS: We developed a functional chimeric TCR against PSCA for treatment of PCa. The chimeric alpha-PSCA-beta2/CD3zeta-TCR might now be used for arming human cytotoxic T-cells for further studies towards a clinical treatment of PCa.

Coffee and Maillard products activate NF-kappaB in macrophages via H2O2 production.

In this study, we investigated the immunomodulatory activity of coffee and Maillard reaction products on macrophages in vitro. Stimulation of macrophages with coffee, but not with raw coffee extract in PBS, led to a 13-fold increased nuclear NF-kappaB translocation. A Maillard reaction mixture (25 mM D-ribose/L-lysine, 30 min at 120 degrees C) increased NF-kappaB translocation 18-fold (in PBS) or six-fold (in medium). MRPs also induced a two-fold increased NF-kappaB translocation in untransfected human embryonic kidney (HEK) cells as well as in HEK cells stably transfected with the receptor for advanced glycation endproducts (RAGE), indicating that the effect was not RAGE mediated. On the other hand, catalase totally abolished coffee- and MRP-induced NF-kappaB translocation. Consequently, up to 366 microM hydrogen peroxide was measured in the coffee preparation and Maillard mixtures used for cell stimulation. Stimulation of macrophages with MRPs did not lead to significantly increased IL-6 or NO release. Thus, it can be concluded that coffee and MRPs induce NF-kappaB translocation in macrophages via the generation of hydrogen peroxide.

Hypochlorite-modified albumin colocalizes with RAGE in the artery wall and promotes MCP-1 expression via the RAGE-Erk1/2 MAP-kinase pathway.

Signal transduction via the endothelial receptor for advanced glycation end products (RAGE) plays a key role in vascular inflammation. Recent observations have shown that the myeloperoxidase-H2O2-chloride system of activated phagocytes is highly up-regulated under inflammatory conditions where hypochlorous acid (HOCl) is formed as the major oxidant. Albumin, an in vivo carrier for myeloperoxidase is highly vulnerable to oxidation and a major representative of circulating advanced oxidized proteins during inflammatory diseases. Immunohistochemical studies performed in the present study revealed marked colocalization of HOCl-modified epitopes with RAGE and albumin in sections of human atheroma, mainly at the endothelial lining. We show that albumin modified with physiologically relevant concentrations of HOCl, added as reagent or generated by the myeloperoxidase-H2O2-chloride system, is a high affinity ligand for RAGE. Albumin, modified by HOCl in the absence of free amino acids/carbohydrates/lipids to exclude formation of AGE-like structures, induced a rapid, RAGE-dependent activation of extracellular signal-regulated kinase 1/2 and up-regulation of the proinflammatory mediator monocyte chemoattractant protein-1. Cellular activation could be blocked either by a specific polyclonal anti-RAGE IgG and/or a specific mitogen-activated protein-kinase kinase inhibitor. The present study demonstrates that HOCl-modified albumin acts as a ligand for RAGE and promotes RAGE-mediated inflammatory complications.

Identification of a naturally processed T cell epitope derived from the glioma-associated protein SOX11.

The development of T cell-based immunotherapies of cancer depends on the identification of tumor-associated antigens capable of eliciting tumor-directed cytotoxic T cell responses. In malignant glioma the number of well-defined target antigens for cytotoxic T lymphocytes (CTLs) is still very limited. Recently, we demonstrated the abundant and specific overexpression of the transcription factor SOX11 in malignant glioma. Here, we describe the SOX11-derived peptide LLRRYNVAKV which is capable of inducing human leukocyte antigen-A*0201-restricted and tumor-reactive CTLs. This novel CTL epitope may serve as an attractive candidate for a T cell-based immunotherapy of glioma.

Autoantibodies to the inhibitor of apoptosis protein survivin in patients with brain tumors.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family and is frequently expressed in cancers, including meningiomas and gliomas. Survivin may be associated with tumor progression and poor prognosis of patients with brain tumors. Using ELISA and immunoblot analysis we asked whether survivin is capable of eliciting a humoral immune response in patients with meningiomas and gliomas. Survivin-specific antibodies were detected in 5 of 42 (11.9%) patients with meningiomas and 3 of 35 (8.6%) patients with malignant gliomas of the WHO grades 3 and 4, but not in healthy controls. Tumors of patients with detectable anti-survivin antibodies demonstrated survivin expression in at least 20% of the tumor cells as assessed by immunohistochemistry. We conclude that patients with meningiomas and malignant gliomas can mount a high-titer IgG immune response against the 'universal' tumor-associated antigen survivin. Anti-survivin antibodies may represent attractive tools for diagnosis and follow-up of brain tumors.

Nuclear localization of Survivin renders HeLa tumor cells more sensitive to apoptosis by induction of p53 and Bax.

Clinical studies have shown that nuclear expression of the inhibitor of apoptosis protein Survivin in tumor cells predicted a favorable prognosis whereas cytosolic-localized protein caused a decreased overall survival. Therefore Survivin's subcellular localization may be important for its anti-apoptotic capacity. To address this question, we investigated localization and function of Survivin in normal human lung fibroblasts (NHLFs) and HeLa tumor cells. NHLFs of early passages expressed Survivin in the nucleus and were highly sensitive to C2 ceramide, which induces the mitochondrial apoptotic pathway. In contrast, NHLFs at higher passages relocated Survivin to the cytosol and became more resistant to C2 ceramide. Blocking nuclear export of Survivin by leptomycin B in HeLa cells increased susceptibility to C2 ceramide. In addition, transduction of HeLa cells with Survivin fused to a nuclear localization signal augmented basal expression levels of p53 and Bax and enhanced sensitivity for intrinsic apoptosis. Those findings suggest that a predominant nuclear localization of Survivin increases the sensitivity for pro-apoptotic stimuli, whereas nuclear export enables Survivin to fulfill its inhibitor of apoptosis function. A therapeutic intervention which holds Survivin in the nucleus of tumor cells might improve cancer therapy.

Tissue-specificity of prostate specific antigens: comparative analysis of transcript levels in prostate and non-prostatic tissues.

Activation of immune defense mechanisms against tumor antigens appears to be a promising therapeutic option for advanced prostate cancer (PCa). Specific immunotherapy critically depends on target antigens that are selectively expressed in the tumorous and optional in the normal prostate tissue in sufficient amounts. Although several prostate antigens have been described and some have already been used in clinical trials, a detailed comparative evaluation of their tissue-specificity and expression levels is still lacking. We determined the transcript levels of eight prostate targets (PSA, PAP, PSCA, PSGR, Prostein, PSMA, AIbZIP, trp-p8) in 16 different tissues by quantitative PCR and calculated a tissue-specificity index (TSI) for each molecule. Besides a preferential expression in prostate for all targets, striking differences in the expression levels and TSI were revealed which may be important for the selection of appropriate antigens for immunotherapy of PCa.

Transcript quantification of Dresden G protein-coupled receptor (D-GPCR) in primary prostate cancer tissue pairs.

Recently, we identified the novel protein D-GPCR (Dresden G protein-coupled receptor) which is selectively overexpressed in human prostate cancer (PCa) and belongs to the subfamily of odorant-like orphan GPCRs. Quantification of D-GPCR transcripts in paired malignant and non-malignant prostate tissues of 106 patients with primary PCa by real-time PCR demonstrated a significant up-regulation of this gene in tumor samples. Furthermore, its expression increases with higher tumor stages and grades. The evaluation of D-GPCR expression as a potential molecular tumor marker was performed by receiver-operating characteristic curve (ROC) analysis resulting in an area under the curve (AUC) value of 0.6452. Hence, the evaluation of D-GPCR as possible additive diagnostic tool and putative therapy target appears promising.

The receptor for advanced glycation end products is highly expressed in the skin and upregulated by advanced glycation end products and tumor necrosis factor-alpha.

Advanced glycation end products (AGEs) form non-enzymatically from reactions of proteins with reducing sugars. In the skin, AGEs were reported to accumulate in dermal elastin and collagens and to interact nonspecifically with the cell membrane of dermal fibroblasts. Therefore, AGEs may influence the process of skin aging. We investigated the presence of the AGE receptor RAGE in skin and the influence of AGEs on receptor expression and the formation of extracellular matrix (ECM). Sections of sun-protected and sun-exposed skin were analyzed with monoclonal antibodies against (RAGE), heat-shock protein 47, factor XIIIa, CD31, and CD45. RAGE was mainly expressed in fibroblasts, dendrocytes, and keratinocytes and to a minor extent in endothelial and mononuclear cells. Human foreskin fibroblasts (HFFs) highly expressed RAGE on the protein and mRNA level when analyzed by quantitative Western blotting and real-time PCR. Incubation of HFFs with the specific RAGE ligand Nepsilon-(carboxymethyl)lysine-modified BSA (CML-BSA) and tumor necrosis factor-alpha resulted in significant upregulation of RAGE expression. CML-BSA induced a mildly profibrogenic pattern, increasing connective tissue growth factor, transforming growth factor-beta (TGF-beta) 1, and procollagen-alpha1(I) mRNA, whereas expression of matrix metalloproteinase (MMP)-1, -2, -3, and -12 was unaffected. We conclude that in HFFs, AGE-RAGE interactions may influence the process of skin aging through mild stimulation of ECM gene expression.

Age-dependent changes of glyoxalase I expression in human brain.

Increased modification and crosslinking of proteins by advanced glycation end products (AGEs) is a characteristic feature of aging, and contributes to the formation of many of the lesions of neurodegenerative diseases including neurofibrillary tangles and amyloid plaques in Alzheimer's disease. Therefore, defense mechanisms against AGE formation or detoxification of their precursors such as the glyoxalase system are of particular interest in aging research. Thus, we investigated the age-dependent protein expression, the activity as well as the RNA level of glyoxalase I in Brodmann area 22 (auditory association area of superior temporal gyrus) of the human cerebral cortex. Our immunohistochemical results demonstrate the localization of glyoxalase I in neurons, predominantly pyramidal cells, as well as in astroglia, located predominantly in the subpial region. The number of glyoxalase I expressing neurons and astroglia increases with age, with a peak at approximately 55 years, and progressively decreases thereafter. These results were confirmed by biochemical investigations in total brain tissue, where the RNA, the protein level as well as the activity of glyoxalase I enzyme were analyzed in different age groups. In conclusion, the increase in glyoxalase I expression up to the age of 55 may be a compensatory mechanism against high oxoaldyde levels and the accumulation of AGEs. However, the decline of glyoxalase expression and activity in old age, possibly caused by impairment in transcription or/and translation, may subsequently lead to increased levels of reactive carbonyl compounds, followed by protein crosslinking, inflammation, oxidative stress and neuronal degeneration.