SARM1 regulates pro-inflammatory cytokine expression in human monocytes by NADase-dependent and -independent mechanisms.

SARM1 is a Toll-IL-1 receptor (TIR) domain-containing protein with roles in innate immunity and neuronal death in diverse organisms. Unlike other innate immune TIR proteins that function as adaptors for Toll-like receptors (TLRs), SARM1 has NADase activity, and this activity regulates murine neuronal cell death. However, whether human SARM1, and its NADase activity, are involved in innate immune regulation remains unclear. Here, we show that human SARM1 regulates proinflammatory cytokine expression in both an NADase-dependent and -independent manner in monocytes. SARM1 negatively regulated TLR4-dependent TNF mRNA induction independently of its NADase activity. In contrast, SARM1 inhibited IL-1ß secretion through both NADase-dependent inhibition of pro-IL-1ß expression, and NADase-independent suppression of the NLRP3 inflammasome and hence processing of pro-IL-1ß to mature IL-1ß. Our study reveals multiple mechanisms whereby SARM1 regulates pro-inflammatory cytokines in human monocytes and shows, compared to other mammalian TIR proteins, a distinct NADase-dependent role for SARM1 in innate immunity.

SARM1 regulates NAD+-linked metabolism and select immune genes in macrophages.

Sterile alpha and HEAT/armadillo motif-containing protein (SARM1) was recently described as a NAD+-consuming enzyme and has previously been shown to regulate immune responses in macrophages. Neuronal SARM1 is known to contribute to axon degeneration due to its NADase activity. However, how SARM1 affects macrophage metabolism has not been explored. Here, we show that macrophages from Sarm1-/- mice display elevated NAD+ concentrations and lower cyclic ADP-ribose, a known product of SARM1-dependent NAD+ catabolism. Further, SARM1-deficient macrophages showed an increase in the reserve capacity of oxidative phosphorylation and glycolysis compared to WT cells. Stimulation of macrophages to a proinflammatory state by lipopolysaccharide (LPS) revealed that SARM1 restricts the ability of macrophages to upregulate glycolysis and limits the expression of the proinflammatory gene interleukin (Il) 1b, but boosts expression of anti-inflammatory Il10. In contrast, we show macrophages lacking SARM1 induced to an anti-inflammatory state by IL-4 stimulation display increased oxidative phosphorylation and glycolysis, and reduced expression of the anti-inflammatory gene, Fizz1. Overall, these data show that SARM1 fine-tunes immune gene transcription in macrophages via consumption of NAD+ and altered macrophage metabolism.

Prenatal exposure to Cannabis smoke induces early and lasting damage to the brain.

Cannabis is the most widely used illegal drug during pregnancy, however, the effects of gestational exposure to Cannabis smoke (CS) on the central nervous system development remain uncharacterised. This study investigates the effects of maternal CS inhalation on brain function in the offspring. Pregnant mice were exposed daily to 5 min of CS during gestational days (GD) 5.5-17.5. On GD 18.5 half of the dams were euthanized for foetus removal. The offspring from the remaining dams were euthanized on postnatal days (PND) 20 and 60 for evaluation. Brain volume, cortex cell number, SOX2, histone-H3, parvalbumin, NeuN, and BDNF immunoreactivity were assessed in all groups. In addition, levels of NeuN, CB1 receptor, and BDNF expression were assessed and cortical primary neurons from rats were treated with Cannabis smoke extract (CSE) for assessment of cell viability. We found that male foetuses from the CS exposed group had decreased brain volume, whereas mice at PND 60 from the exposed group presented with increased brain volume. Olfactory bulb and diencephalon volume were found lower in foetuses exposed to CS. Mice at PND 60 from the exposed group had a smaller volume in the thalamus and hypothalamus while the cerebellum presented with a greater volume. Also, there was an increase in cortical BDNF immunoreactivity in CS exposed mice at PND 60. Protein expression analysis showed an increase in pro-BDNF in foetus brains exposed to CS. Mice at PND 60 presented an increase in mature BDNF in the prefrontal cortex (PFC) in the exposed group and a higher CB1 receptor expression in the PFC. Moreover, hippocampal NeuN expression was higher in adult animals from the exposed group. Lastly, treatment of cortical primary neurons with doses of CSE resulted in decreased cell viability. These findings highlight the potential negative neurodevelopmental outcomes induced by gestational CS exposure.

In silico analysis of the human milk oligosaccharide glycome reveals key enzymes of their biosynthesis.

Human milk oligosaccharides (HMOs) form the third most abundant component of human milk and are known to convey several benefits to the neonate, including protection from viral and bacterial pathogens, training of the immune system, and influencing the gut microbiome. As HMO production during lactation is driven by enzymes that are common to other glycosylation processes, we adapted a model of mucin-type GalNAc-linked glycosylation enzymes to act on free lactose. We identified a subset of 11 enzyme activities that can account for 206 of 226 distinct HMOs isolated from human milk and constructed a biosynthetic reaction network that identifies 5 new core HMO structures. A comparison of monosaccharide compositions demonstrated that the model was able to discriminate between two possible groups of intermediates between major subnetworks, and to assign possible structures to several previously uncharacterised HMOs. The effect of enzyme knockouts is presented, identifying ß-1,4-galactosyltransferase and ß-1,3-N-acetylglucosaminyltransferase as key enzyme activities involved in the generation of the observed HMO glycosylation patterns. The model also provides a synthesis chassis for the most common HMOs found in lactating mothers.

O-Glycologue: A Formal-Language-Based Generator of O-Glycosylation Networks.

The web application O-Glycologue provides an online simulation of the biosynthetic enzymes of O-linked glycosylation, using a knowledge-based system described previously. Glycans can be imported in GlycoCT condensed format, or else as IUPAC condensed names, and passed as substrates to the enzymes, which are modeled as regular-expression-based substitutions on strings. The resulting networks of reactions can be exported as SBML. The effects of knocking out different sets of enzyme activities can be compared. A method is provided for predicting the enzymes required to produce a given substrate, using an O-glycan from human gastric mucin as an example. The system has been adapted to other systems of glycosylation enzymes, and an application to ganglioside oligosaccharide synthesis is demonstrated. O-Glycologue is available at https://glycologue.org/o/ .

Metabolic Labeling of Primary Neurons Using Carbohydrate Click Chemistry.

Glycans play an important role in many neuronal processes, such as neurotransmitter release and reuptake, cell-cell communication and adhesion, modulation of ion channel activity, and immune function. Carbohydrate click chemistry is a powerful technique for studying glycan function and dynamics in vitro, in vivo, and ex vivo. Here, we use commercially available synthetic tetraacetylated azido sugars, copper and copper-free click chemistry to metabolically label and analyze primary rat cortical neurons. In addition, we use high resolution confocal and STED microscopy to image and analyze different forms of glycosylation in ultrahigh resolution. We observe different patterns of GlcNAz, GalNAz, and ManNAz distribution at different stages of neuronal development. We also observe highly sialylated structures on the neuronal plasma membrane, which warrant further investigation.

Monitoring the Sialome on Human Immune Cells.

The sialome or display of sialic acids on the surface of human immune cells can vary according to immune response and activation state. Here, human peripheral blood mononuclear cells (PBMCs) were isolated and activated with anti-CD3 antibody and the cell surface sialome was quantified using a combination of click chemistry, confocal microscopy and flow cytometry techniques. Carbohydrate click chemistry was used to detect and measure the incorporation of an azido-m65odified sialic acid precursor molecule, N-acetylmannosamine (ManNaz) sugar into the PBMC surface sialome. Incorporation of sialic acid into the PBMC glycocalyx was visualized using copper-catalyzed click conjugation of Alexa 488 alkyne and confocal microscopy and further quantified using flow cytometry. The use of these methods indicate that regulating the sialome content on the surface of activated immune cells may be monitored during immunomodulatory responses and anti-inflammatory therapies.

Simulating the enzymes of ganglioside biosynthesis with Glycologue.

Gangliosides are an important class of sialylated glycosphingolipids linked to ceramide that are a component of the mammalian cell surface, especially those of the central nervous system, where they function in intercellular recognition and communication. We describe an in silico method for determining the metabolic pathways leading to the most common gangliosides, based on the known enzymes of their biosynthesis. A network of 41 glycolipids is produced by the actions of the 10 enzymes included in the model. The different ganglioside nomenclature systems in common use are compared and a systematic variant of the widely used Svennerholm nomenclature is described. Knockouts of specific enzyme activities are used to simulate congenital defects in ganglioside biosynthesis, and altered ganglioside status in cancer, and the effects on network structure are predicted. The simulator is available at the Glycologue website, https://glycologue.org/.

Mitochondrial arginase-2 is essential for IL-10 metabolic reprogramming of inflammatory macrophages.

Mitochondria are important regulators of macrophage polarisation. Here, we show that arginase-2 (Arg2) is a microRNA-155 (miR-155) and interleukin-10 (IL-10) regulated protein localized at the mitochondria in inflammatory macrophages, and is critical for IL-10-induced modulation of mitochondrial dynamics and oxidative respiration. Mechanistically, the catalytic activity and presence of Arg2 at the mitochondria is crucial for oxidative phosphorylation. We further show that Arg2 mediates this process by increasing the activity of complex II (succinate dehydrogenase). Moreover, Arg2 is essential for IL-10-mediated downregulation of the inflammatory mediators succinate, hypoxia inducible factor 1α (HIF-1α) and IL-1ß in vitro. Accordingly, HIF-1α and IL-1ß are highly expressed in an LPS-induced in vivo model of acute inflammation using Arg2-/- mice. These findings shed light on a new arm of IL-10-mediated metabolic regulation, working to resolve the inflammatory status of the cell.

Complex I Controls Mitochondrial and Plasma Membrane Potentials in Nerve Terminals.

Reductions in the activities of mitochondrial electron transport chain (ETC) enzymes have been implicated in the pathogenesis of numerous chronic neurodegenerative disorders. Maintenance of the mitochondrial membrane potential (Δψm) is a primary function of these enzyme complexes, and is essential for ATP production and neuronal survival. We examined the effects of inhibition of mitochondrial ETC complexes I, II/III, III and IV activities by titrations of respective inhibitors on Δψm in synaptosomal mitochondria. Small perturbations in the activity of complex I, brought about by low concentrations of rotenone (1-50 nM), caused depolarisation of Δψm. Small decreases in complex I activity caused an immediate and partial Δψm depolarisation, whereas inhibition of complex II/III activity by more than 70% with antimycin A was required to affect Δψm. A similarly high threshold of inhibition was found when complex III was inhibited with myxothiazol, and inhibition of complex IV by more than 90% with KCN was required. The plasma membrane potential (Δψp) had a complex I inhibition threshold of 40% whereas complex III and IV had to be inhibited by more than 90% before changes in Δψp were registered. These data indicate that in synaptosomes, both Δψm and Δψp are more susceptible to reductions in complex I activity than reductions in the other ETC complexes. These findings may be of relevance to the mechanism of neuronal cell death in Parkinson's disease in particular, where such reductions in complex I activity are present.

ß-Naphthoflavone and Ethanol Reverse Mitochondrial Dysfunction in A Parkinsonian Model of Neurodegeneration.

The 1-methyl-4-phenylpyridinium (MPP+) is a parkinsonian-inducing toxin that promotes neurodegeneration of dopaminergic cells by directly targeting complex I of mitochondria. Recently, it was reported that some Cytochrome P450 (CYP) isoforms, such as CYP 2D6 or 2E1, may be involved in the development of this neurodegenerative disease. In order to study a possible role for CYP induction in neurorepair, we designed an in vitro model where undifferentiated neuroblastoma SH-SY5Y cells were treated with the CYP inducers ß-naphthoflavone (ßNF) and ethanol (EtOH) before and during exposure to the parkinsonian neurotoxin, MPP+. The toxic effect of MPP+ in cell viability was rescued with both ßNF and EtOH treatments. We also report that this was due to a decrease in reactive oxygen species (ROS) production, restoration of mitochondrial fusion kinetics, and mitochondrial membrane potential. These treatments also protected complex I activity against the inhibitory effects caused by MPP+, suggesting a possible neuroprotective role for CYP inducers. These results bring new insights into the possible role of CYP isoenzymes in xenobiotic clearance and central nervous system homeostasis.

Degradation of thymic humoral factor γ2 in human, rat and mouse blood: An experimental and theoretical study.

The degradation of the immunomodulatory octapeptide, thymic humoral factor γ2 (THF-γ2, thymoctonan) has been studied in whole blood samples from human, rat and mouse. The peptide, Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu, was shown to be rapidly degraded by peptidases. The half-life of the intact peptide was less than 6 min at 37 °C in blood from the three species tested. The main fragments formed from THF-γ2 were found to be Glu-Asp-Gly-Pro-Lys-Phe-Leu (2-8), Asp-Gly-Pro-Lys-Phe-Leu (3-8) and Glu-Asp-Gly-Pro-Lys (2-6) in human and in rat blood and 2-8 and 2-6 in mouse blood. Analysis of the time course of degradation revealed a sequential removal of single amino acids from the N-terminus (aminopeptidase activities) in a process that was apparently unable to cleave the Gly-Pro bond (positions 4-5 in the peptide) together with an independent cleavage of the Lys-Phe bond (positions 6-7 in the peptide) to release the dipeptide Phe-Leu. This behaviour and the effects of inhibitors showed the involvement of metallo-exopeptidases in the N-terminal digestion and a phosphoramidon-sensitive metallo-endopeptidase in the cleavage of the Lys-Phe bond. The degradation patterns in human blood were modelled in terms of the competing pathways involved approximating to first-order kinetics, and an analytical solution obtained via the method of Laplace Transforms. The half-life of THF degradation in whole rat blood sample was found to be significantly lower than in human or mouse.

6-Hydroxydopamine: a far from simple neurotoxin.

6-Hydroxydopamine (6-OHDA), which is a neurotoxin that selectively destroys catecholaminergic nerves in sympathetically innervated tissues, has been used to provide a model of Parkinson's disease in experimental animals. It is rapidly autoxidised to yield potentially toxic products and reactive oxygen species. Its ability to release Fe(II) from protein storage sites also results in the formation of hROS. This account will consider how this family of toxic products may contribute to the observed effects of 6-OHDA.

Dichloroacetate Stabilizes Mitochondrial Fusion Dynamics in Models of Neurodegeneration.

Mitochondrial dysfunction is a recognized hallmark of neurodegenerative diseases and abnormal mitochondrial fusion-fission dynamics have been implicated in the pathogenesis of neurodegenerative disorders. This study characterizes the effects of metabolic flux inhibitors and activators on mitochondrial fusion dynamics in the neuronal cell culture model of differentiated PC12 cells. Using a real time confocal microscopy assay, it was found that the carnitine palmitoyltransferase I (CPTI) inhibitor, etomoxir, reduced mitochondrial fusion dynamics in a time-dependent manner. Etomoxir also decreased JO2, ΔΨm and reactive oxygen species (ROS) production rates. The mitochondrial pyruvate carrier (MPC) inhibitor, UK5099, reduced fusion dynamics and in combination with etomoxir these inhibitory effects were amplified. Use of the pyruvate dehydrogenase (PDH) kinase inhibitor dichloroacetate, which is known to increase metabolic flux through PDH, reversed the etomoxir-induced effects on fusion dynamics, JO2, ΔΨm but not ROS production rates. Dichloroacetate also partially reversed inhibition of mitochondrial fusion dynamics caused by the parkinsonian-inducing neurotoxin, MPP+. These results suggest that dichloroacetate-induced activation of metabolic flux in the mitochondrion may be a mechanism to restore normal mitochondrial fusion-fission dynamics in metabolically challenged cells.

Salivary N-glycosylation as a biomarker of oral cancer: A pilot study.

Reliable biomarkers for oral cancer (OC) remain scarce, and routine tests for the detection of precancerous lesions are not routine in the clinical setting. This study addresses a current unmet need for more sensitive and quantitative tools for the management of OC. Whole saliva was used to identify and characterize the nature of glycans present in saliva and determine their potential as OC biomarkers. Proteins obtained from whole saliva were subjected to PNGase F enzymatic digestion. The resulting N-glycans were analyzed with weak anion exchange chromatography, exoglycosidase digestions coupled to ultra-high performance liquid chromatography and/or mass spectrometry. To determine N-glycan changes, 23 individuals with or without cancerous oral lesions were analyzed using Hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC), and peak-based area relative quantitation was performed. An abundant and complex salivary N-glycomic profile was identified. The main structures present in saliva were neutral oligosaccharides consisting of high mannose, hybrid and complex structures, followed by smaller fractions of mono and di-sialylated structures. To determine if differential N-glycosylation patterns distinguish between OC and control groups, Mann-Whitney testing and principle component analysis (PCA) were used. Eleven peaks were shown to be statistically significant (P ≤ 0.05), while PCA analysis showed segregation of the two groups based on their glycan profile. N-glycosylation changes are active in the oral carcinogenic process and may serve as biomarkers for early detection to reduce morbidity and mortality. Identifying which N-glycans contribute most in the carcinogenic process may lead to their use in the detection, prognosis and treatment of OC.

A mechanism for bistability in glycosylation.

Glycosyltransferases are a class of enzymes that catalyse the posttranslational modification of proteins to produce a large number of glycoconjugate acceptors from a limited number of nucleotide-sugar donors. The products of one glycosyltransferase can be the substrates of several other enzymes, causing a combinatorial explosion in the number of possible glycan products. The kinetic behaviour of systems where multiple acceptor substrates compete for a single enzyme is presented, and the case in which high concentrations of an acceptor substrate are inhibitory as a result of abortive complex formation, is shown to result in non-Michaelian kinetics that can lead to bistability in an open system. A kinetic mechanism is proposed that is consistent with the available experimental evidence and provides a possible explanation for conflicting observations on the ß-1,4-galactosyltransferases. Abrupt switching between steady states in networks of glycosyltransferase-catalysed reactions may account for the observed changes in glycosyl-epitopes in cancer cells.

Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab.

Fc gamma receptors (FcγR) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of FcγRs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the FcγR glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fcγ receptors RI (CD64), RIIaArg131/His131 (CD32a), RIIb (CD32b) and RIIIaPhe158/Val158 (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each FcγR were characterized and we discuss the CHO-FcγRIIIaPhe158/Val158 and CHO-FcγRI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent FcγR glycosylation. The glycan profiles of CHO expressed FcγRI and FcγRIIIaPhe158/Val158 were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective FcγR binding patterns with rituximab. Oligomannose structures are prevalent on FcγRI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On FcγRI and FcγRIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-FcγR binding kinetics differ depending on the glycosylation of the FcγR and further support a stabilizing role of FcγR glycans in the antibody binding interaction.

Fc gamma receptors: glycobiology and therapeutic prospects.

Therapeutic antibodies hold great promise for the treatment of cancer and autoimmune diseases, and developments in antibody-drug conjugates and bispecific antibodies continue to enhance treatment options for patients. Immunoglobulin (Ig) G antibodies are proteins with complex modifications, which have a significant impact on their function. The most important of these modifications is glycosylation, the addition of conserved glycans to the antibody Fc region, which is critical for its interaction with the immune system and induction of effector activities such as antibody-dependent cell cytotoxicity, complement activation and phagocytosis. Communication of IgG antibodies with the immune system is controlled and mediated by Fc gamma receptors (FcγRs), membrane-bound proteins, which relay the information sensed and gathered by antibodies to the immune system. These receptors are also glycoproteins and provide a link between the innate and adaptive immune systems. Recent information suggests that this receptor glycan modification is also important for the interaction with antibodies and downstream immune response. In this study, the current knowledge on FcγR glycosylation is discussed, and some insight into its role and influence on the interaction properties with IgG, particularly in the context of biotherapeutics, is provided. For the purpose of this study, other Fc receptors such as FcαR, FcεR or FcRn are not discussed extensively, as IgG-based antibodies are currently the only therapeutic antibody-based products on the market. In addition, FcγRs as therapeutics and therapeutic targets are discussed, and insight into and comment on the therapeutic aspects of receptor glycosylation are provided.

Metabolic flux control in glycosylation.

Glycosylation is a common post-translational protein modification, in which glycans are built onto proteins through the sequential addition of monosaccharide units, in reactions catalysed by glycosyltransferases. Glycosylation influences the physicochemical and biological properties of proteins, with subsequent effects on subcellular and extracellular protein trafficking, cell-cell recognition, and ligand-receptor interactions. Glycan structures can be complex, as is the regulation of their biosynthesis, and it is only recently that the systems biology of metabolic flux control and glycosyltransferase networks has become a study in its own right. We review various models of glycosylation that have been proposed to date, based on current knowledge of Golgi structure and function, and consider how metabolic flux through glycosyltransferase networks regulates glycosylation events in the cell.

A Knowledge-Based System for Display and Prediction of O-Glycosylation Network Behaviour in Response to Enzyme Knockouts.

O-linked glycosylation is an important post-translational modification of mucin-type protein, changes to which are important biomarkers of cancer. For this study of the enzymes of O-glycosylation, we developed a shorthand notation for representing GalNAc-linked oligosaccharides, a method for their graphical interpretation, and a pattern-matching algorithm that generates networks of enzyme-catalysed reactions. Software for generating glycans from the enzyme activities is presented, and is also available online. The degree distributions of the resulting enzyme-reaction networks were found to be Poisson in nature. Simple graph-theoretic measures were used to characterise the resulting reaction networks. From a study of in-silico single-enzyme knockouts of each of 25 enzymes known to be involved in mucin O-glycan biosynthesis, six of them, ß-1,4-galactosyltransferase (ß4Gal-T4), four glycosyltransferases and one sulfotransferase, play the dominant role in determining O-glycan heterogeneity. In the absence of ß4Gal-T4, all Lewis X, sialyl-Lewis X, Lewis Y and Sda/Cad glycoforms were eliminated, in contrast to knockouts of the N-acetylglucosaminyltransferases, which did not affect the relative abundances of O-glycans expressing these epitopes. A set of 244 experimentally determined mucin-type O-glycans obtained from the literature was used to validate the method, which was able to predict up to 98% of the most common structures obtained from human and engineered CHO cell glycoforms.