Neuroprotective Effects of Polysialic Acid and SIGLEC-11 in Activated Phagocytic Cells

SIGLEC-11 is an inhibitory receptor expressed on microglial cells and macrophages and can recognize α 2→8 linked Sias structures. The surface of neuron is decorated by different lengths of polySias. PolySia-SIGLEC-11 interaction is important to keep normal physiological conditions in neuron-microglia co-culture systems. However, till now it was not clear which length of polySia is recognized by SIGLEC-11. In this study the low molecular weight polySia with average degree of polymerization 20 (polySia avDP20), among different polySia lengths, introduced as the best length which was recognized by SIGLEC-11. PolySia avDP20 pre-treatment upon Aβ or debris stimulation kept superoxide release of microglia/macrophages as low as of untreated cells. This effect was not observed when cells were pre-treated with monoSia or oligoSias. Furthermore, compared to other polySia lengths (avDP60 and avDP180), polySia avDP20 had no effect on the metabolic activity of cells. Knockdown of SIGLEC-11 was enough to prevent the inhibitory function of polySia avDP20. Additional experiments showed that the anti-superoxide effect of polySia avDP20 was as potent as Trolox and SOD1. Phagocytosis analysis in iPSdM cells and macrophages revealed that polySia avDP20 pre-treatment did reduce uptake of Aβ and debris, which are inflammatory phagocytosis stimulants. Neurons were differentiated from pNSCs to investigate the consequence of polySia avDP20 addition to co-cultures with iPSdM/macrophages. Co-culture of Aβ or LPS stimulated iPSdM/macrophage with neurons led to shorter neurite length. This length could stay like untreated neurons if polySia avDP20 was present. Thus, this study suggests polySia avDP20 as a ligand for SIGLEC-11 receptor to reduce the inflammatory response of phagocytes towards provoking stimulants

Phagocytosis‐related NADPH oxidase 2 subunit gp91phox contributes to neurodegeneration after repeated systemic challenge with lipopolysaccharides

Repeated systemic challenge with lipopolysaccharides (LPS) can induce microglia activation and inflammatory neurodegeneration in the substantia nigra pars compacta region of mice. We now explored the role of mononuclear phagocytes associated nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX‐2) in inflammatory neurodegeneration. Cybb ‐deficient NOX‐2 knock‐out (KO) and control wild type (WT) mice were treated intraperitoneally daily over four consecutive days with 1 μg/gbw/day LPS. Transcriptome analysis by RNA‐seq of total brain tissue indicated increased LPS‐induced upregulation of genes belonging to the reactive oxygen species and reactive nitrogen species production, complement and lysosome activation as well as apoptosis and necroptosis in WT compared to NOX‐2 KO mice. Validation of up‐regulated gene transcripts via qRT‐PCR confirmed that LPS‐challenged NOX‐2 KO mice expressed lower levels of the microglial phagocytosis‐related genes Nos2 , Cd68 , Aif1/Iba1 , Cyba , Itgam , and Fcer1g compared to WT mice at Day 5 after systemic inflammatory challenge, but no significant differences in the pro‐inflammatory genes Tnfα and Il1b as well as microglial IBA1 and CD68 intensities were observed between both genotypes. Furthermore, loss of tyrosine hydroxylase positive (TH+) and NeuN positive neurons in the substantia nigra pars compacta upon repeated systemic LPS application were attenuated in NOX‐2 KO mice. Thus, our data demonstrate that loss of dopaminergic neurons in the substantia nigra pars compacta after repeated systemic challenge with LPS is associated with a microglial phagocytosis‐related gene activation profile involving the NADPH oxidase subunit Cybb/gp91phox

Anti-inflammatory activity of low molecular weight polysialic acid on human macrophages

Oligosialic and polysialic acid (oligoSia and polySia) of the glycocalyx of neural and immune cells are linear chains, in which the sialic acid monomers are alpha 2.8-glycosidically linked. Sialic acid-binding immunoglobulin-like lectin-11 (SIGLEC-11) is a primate-lineage specific receptor of human tissue macrophages and microglia that binds to alpha 2.8-linked oligoSia. Here, we show that soluble low molecular weight polySia with an average degree of polymerization 20 (avDP20) interacts with SIGLEC-11 and acts anti-inflammatory on human THP1 macrophages involving the SIGLEC-11 receptor. Soluble polySia avDP20 inhibited the lipopolysaccharide (LPS)-induced gene transcription and protein expression of tumor necrosis factor-alpha (Tumor Necrosis Factor Superfamily Member 2, TNFSF2). In addition, polySia avDP20 neutralized the LPS-triggered increase in macrophage phagocytosis, but did not affect basal phagocytosis or endocytosis. Moreover, polySia avDP20 prevented the oxidative burst of human macrophages triggered by neural debris or fibrillary amyloid-beta(1-42). In a human macrophage-neuron co-culture system, polySia avDP20 also reduced loss of neurites triggered by fibrillary amyloid-beta(1-42). Thus, treatment with polySia avDP20 might be a new anti-inflammatory therapeutic strategy that also prevents the oxidative burst of macrophages

Polysialic acid blocks mononuclear phagocyte reactivity, inhibits complement activation, and protects from vascular damage in the retina

Age-related macular degeneration (AMD) is a major cause of blindness in the elderly population. Its pathophysiology is linked to reactive oxygen species (ROS) and activation of the complement system. Sialic acid polymers prevent ROS production of human mononuclear phagocytes via the inhibitory sialic acid-binding immunoglobulin-like lectin-11 (SIGLEC11) receptor. Here, we show that low-dose intravitreal injection of low molecular weight polysialic acid with average degree of polymerization 20 (polySia avDP20) in humanized transgenic mice expressing SIGLEC11 on mononuclear phagocytes reduced their reactivity and vascular leakage induced by laser coagulation. Furthermore, polySia avDP20 prevented deposition of the membrane attack complex in both SIGLEC11 transgenic and wild-type animals. In vitro, polySia avDP20 showed two independent, but synergistic effects on the innate immune system. First, polySia avDP20 prevented tumor necrosis factor-, vascular endothelial growth factor A, and superoxide production by SIGLEC11-positive phagocytes. Second, polySia avDP20 directly interfered with complement activation. Our data provide evidence that polySia avDP20 ameliorates laser-induced damage in the retina and thus is a promising candidate to prevent AMD-related inflammation and angiogenesis