Soluble mannose receptor induces proinflammatory macrophage activation and metaflammation.

Proinflammatory activation of macrophages in metabolic tissues is critically important in the induction of obesity-induced metaflammation. Here, we demonstrate that the soluble mannose receptor (sMR) plays a direct functional role in both macrophage activation and metaflammation. We show that sMR binds CD45 on macrophages and inhibits its phosphatase activity, leading to an Src/Akt/NF-κB-mediated cellular reprogramming toward an inflammatory phenotype both in vitro and in vivo. Remarkably, increased serum sMR levels were observed in obese mice and humans and directly correlated with body weight. Importantly, enhanced sMR levels increase serum proinflammatory cytokines, activate tissue macrophages, and promote insulin resistance. Altogether, our results reveal sMR as regulator of proinflammatory macrophage activation, which could constitute a therapeutic target for metaflammation and other hyperinflammatory diseases.

Prevalence of viral hepatitis B and C in Sierra Leone-current knowledge and knowledge gaps: a narrative review.

There are no comprehensive data on viral hepatitis from Sierra Leone; however, a huge disease burden has been observed in different subpopulations. This review summarizes available data on hepatitis B and C virus (HBV and HCV) prevalence in Sierra Leone and identifies knowledge gaps. Despite the non-uniformity of the studies and the lack of systematic case recording, different reports published in recent decades yielded a hepatitis B prevalence of 8.7% among healthcare workers, 11.3% among pregnant women, 15.2% among blood donors and 16.7% in school-age children. The actual HBV prevalence in the general population was reported as 21.7%; similar to what was reported for people living with human immunodeficiency virus (PLHIV). HCV prevalence is 8% and 7% in male and female blood donors, respectively, 4.1% in PLHIV and 2.0% in school children. There are significant knowledge gaps regarding the prevalence of viral hepatitis B and C in Sierra Leone, despite the high burden reported in a few studies. There are limited programmatic interventions on the control and prevention of viral hepatitis in the country. Therefore, well-structured representative studies should provide a solid understanding of the true prevalence of hepatitis B and C to inform best possible public health measures in Sierra Leone.

The helminth glycoprotein omega-1 improves metabolic homeostasis in obese mice through type 2 immunity-independent inhibition of food intake.

Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet, a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega-1 (ω1), one of the major S mansoni immunomodulatory glycoproteins, on metabolic homeostasis. We show that treatment of obese mice with plant-produced recombinant ω1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass, and improved systemic insulin sensitivity and glucose tolerance in a time- and dose-dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils, and alternatively activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of ω1 were still observed in obese STAT6-deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that ω1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole-body energy expenditure, an effect also occurring in leptin receptor-deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein ω1 can induce type 2 immunity, it improves whole-body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6-independent mechanism.

Schistosoma haematobium infection is associated with lower serum cholesterol levels and improved lipid profile in overweight/obese individuals.

Infection with parasitic helminths has been reported to improve insulin sensitivity and glucose homeostasis, lowering the risk for type 2 diabetes. However, little is known about its impact on whole-body lipid homeostasis, especially in obese individuals. For this purpose, a cross-sectional study was carried out in lean and overweight/obese adults residing in the Lambaréné region of Gabon, an area endemic for Schistosoma haematobium. Helminth infection status, peripheral blood immune cell counts, and serum metabolic and lipid/lipoprotein levels were analyzed. We found that urine S. haematobium egg-positive individuals exhibited lower serum total cholesterol (TC; 4.42 vs 4.01 mmol/L, adjusted mean difference [95%CI] -0.30 [-0.68,-0.06]; P = 0.109), high-density lipoprotein (HDL)-C (1.44 vs 1.12 mmol/L, -0.24 [-0.43,-0.06]; P = 0.009) and triglyceride (TG; 0.93 vs 0.72 mmol/L, -0.20 [-0.39,-0.03]; P = 0.022) levels than egg-negative individuals. However, when stratified according to body mass index, these effects were only observed in overweight/obese infected individuals. Similarly, significant negative correlations between the intensity of infection, assessed by serum circulating anodic antigen (CAA) concentrations, and TC (r = -0.555; P<0.001), HDL-C (r = -0.327; P = 0.068), LDL-C (r = -0.396; P = 0.025) and TG (r = -0.381; P = 0.032) levels were found in overweight/obese individuals but not in lean subjects. Quantitative lipidomic analysis showed that circulating levels of some lipid species associated with cholesterol-rich lipoprotein particles were also significantly reduced in overweight/obese infected individuals in an intensity-dependent manner. In conclusion, we reported that infection with S. haematobium is associated with improved lipid profile in overweight/obese individuals, a feature that might contribute reducing the risk of cardiometabolic diseases in such population.

Determination of Adenine Nucleotide Concentrations in Cells and Tissues by High-Performance Liquid Chromatography.

The serine/threonine AMP-activated protein kinase (AMPK) is a central player in the regulation of energy homeostasis, and its activity is tightly controlled, among other mechanisms, by subtle changes in cellular adenine nucleotide levels. In this chapter, we describe a step-by-step protocol for rapid, highly sensitive, reproducible, and simultaneous determination of ATP, ADP, and AMP concentrations in cell or tissue samples by reversed-phase high-performance liquid chromatography (HPLC).

Chronic helminth infection and helminth-derived egg antigens promote adipose tissue M2 macrophages and improve insulin sensitivity in obese mice.

Chronic low-grade inflammation associated with obesity contributes to insulin resistance and type 2 diabetes. Helminth parasites are the strongest natural inducers of type 2 immune responses, and short-lived infection with rodent nematodes was reported to improve glucose tolerance in obese mice. Here, we investigated the effects of chronic infection (12 weeks) with Schistosoma mansoni, a helminth that infects millions of humans worldwide, on whole-body metabolic homeostasis and white adipose tissue (WAT) immune cell composition in high-fat diet-induced obese C57BL/6 male mice. Our data indicate that chronic helminth infection reduced body weight gain (-62%), fat mass gain (-89%), and adipocyte size; lowered whole-body insulin resistance (-23%) and glucose intolerance (-16%); and improved peripheral glucose uptake (+25%) and WAT insulin sensitivity. Analysis of immune cell composition by flow cytometry and quantitative PCR (qPCR) revealed that S. mansoni promoted strong increases in WAT eosinophils and alternatively activated (M2) macrophages. Importantly, injections with S. mansoni-soluble egg antigens (SEA) recapitulated the beneficial effect of parasite infection on whole-body metabolic homeostasis and induced type 2 immune responses in WAT and liver. Taken together, we provide novel data suggesting that chronic helminth infection and helminth-derived molecules protect against metabolic disorders by promoting a T helper 2 (Th2) response, eosinophilia, and WAT M2 polarization.

A novel dominant hyperekplexia mutation Y705C alters trafficking and biochemical properties of the presynaptic glycine transporter GlyT2.

Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this orphan disorder can have serious consequences, including sudden infant death. Dominant and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1) are the major cause of this disorder. However, recessive mutations in the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of startle disease. In this study, systematic DNA sequencing of SLC6A5 revealed a new dominant GlyT2 mutation: pY705C (c.2114A→G) in transmembrane domain 11, in eight individuals from Spain and the United Kingdom. Curiously, individuals harboring this mutation show significant variation in clinical presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal respiration, facial dysmorphism, delayed motor development, or intellectual disability. We functionally characterized this mutation using molecular modeling, electrophysiology, [(3)H]glycine transport, cell surface expression, and cysteine labeling assays. We found that the introduced cysteine interacts with the cysteine pair Cys-311-Cys-320 in the second external loop of GlyT2. This interaction impairs transporter maturation through the secretory pathway, reduces surface expression, and inhibits transport function. Additionally, Y705C presents altered H(+) and Zn(2+) dependence of glycine transport that may affect the function of glycinergic neurotransmission in vivo.

Cell surface turnover of the glutamate transporter GLT-1 is mediated by ubiquitination/deubiquitination.

The main glutamate transporter in the brain, GLT-1, mediates glutamatergic neurotransmission in both physiological and pathological conditions. GLT-1 activity is controlled by both constitutive and regulated trafficking, and although recent evidence indicates that the turnover of this protein in the plasma membrane is accelerated by protein kinase C via an ubiquitin-dependent process, the mechanisms driving the constitutive trafficking of GLT-1 remain unexplored. Here, we used a heterologous system and primary astrocytes to investigate the turnover of GLT-1 and the role of ubiquitin attachment in this process. We show that GLT-1 is endocytosed constitutively in a clathrin-dependent manner, recycling the transporter into endosomes containing EEA1 and Rab4, a marker of rapidly recycling endosomes, and not Rab11 or Rab7, markers of the slow recycling and late endosomal compartments, respectively. We also show that this process is dependent on ubiquitination, because the inhibitor of the ubiquitin-activating enzyme E1, 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester, promotes the retention of GLT-1 at the plasma membrane. Moreover, site-directed mutagenesis demonstrated the involvement of lysines 517 and 526 of GLT-1 in the constitutive internalization of the transporter. The translocation of GLT-1 from the recycling endosomes to the plasma membrane was blocked by LDN-57444, a specific inhibitor to the deubiquitinating enzyme (DUB) ubiquitin C-terminal hydrolase-L1, but not by an inhibitor of the related DUB ubiquitin C-terminal hydrolase-L3, supporting the existence of specific ubiquitination/deubiquitination cycles that ensure the correct concentrations of GLT-1 at the cell surface.

Protein kinase C (PKC)-promoted endocytosis of glutamate transporter GLT-1 requires ubiquitin ligase Nedd4-2-dependent ubiquitination but not phosphorylation.

Glutamate transporter-1 (GLT-1) is the main glutamate transporter in the central nervous system, and its concentration severely decreases in neurodegenerative diseases. The number of transporters in the plasma membrane reflects the balance between their insertion and removal, and it has been reported that the regulated endocytosis of GLT-1 depends on its ubiquitination triggered by protein kinase C (PKC) activation. Here, we identified serine 520 of GLT-1 as the primary target for PKC-dependent phosphorylation, although elimination of this serine did not impair either GLT-1 ubiquitination or endocytosis in response to phorbol esters. In fact, we present evidence indicating that the ubiquitin ligase Nedd4-2 mediates the PKC-dependent ubiquitination and down-regulation of GLT-1. Overexpression of Nedd4-2 increased the ubiquitination of the transporter and promoted its degradation. Moreover, phorbol myristate acetate enhanced Nedd4-2 phosphorylation and the formation of GLT-1·Nedd4-2 complexes, whereas siRNA knockdown of Nedd4-2 prevented ubiquitination, endocytosis, and the concomitant decrease in GLT-1 activity triggered by PKC activation. These results indicate that GLT-1 endocytosis is independent of its phosphorylation and that Nedd4-2 mediates PKC-dependent down-regulation of the transporter.

Splice variants of the glutamate transporter GLT1 form hetero-oligomers that interact with PSD-95 and NMDA receptors.

The glutamate transporter GLT1 is expressed in at least two isoforms, GLT1a and GLT1b, which differ in their C termini. As GLT1 is an oligomeric protein, we have investigated whether GLT1a and GLT1b might associate as hetero-oligomers. Differential tagging (HA-GLT1a and YFP-GLT1b) revealed that these isoforms form complexes that could be immunoprecipitated when co-expressed in heterologous systems. The association of GLT1a and GLT1b was also observed in mixed primary cultures of rat brain and in the adult rat brain, where specific antibodies for GLT1a immunoprecipitated GLT1b and vice versa. Dual immunofluorescence in mixed cultures demonstrated the partial co-localization of both isoforms in neurons and in glial cells. Because GLT1b interacts with an organizer of post-synaptic densities, PSD-95, we examined the capacity of GLT1a to associate with this protein. GLT1a was immunoprecipitated from the rat brain in protein complexes that contained not only GLT1b but also PSD-95 and NMDAR. The interaction between GLT1a with PSD-95 and NMDAR was reproduced in transfected COS7 cells and it appears to be indirect as it requires the presence of GLT1b. These results indicate that the major isoform of the glutamate transporter, GLT1a, can acquire the capacity to interact with PDZ proteins through its inclusion in hetero-oligomers containing GLT1b.

PKC-dependent endocytosis of the GLT1 glutamate transporter depends on ubiquitylation of lysines located in a C-terminal cluster.

The activity of the main glutamate transporter in the CNS, GLT1, can be regulated by protein kinase C (PKC). It is known that activation of PKC by phorbol esters promotes the clathrin-dependent internalization of the transporter, followed by its lysosomal degradation. However, the molecular mechanisms that link PKC activation and the internalization of GLT1 are not fully understood. In this article, we show that this internalization process is dependent on the ubiquitylation of lysine residues located in the C-terminal tail of GLT1. Exposure to PMA increases the ubiquitylation of GLT1 in transfected cells and in the rat brain, and this ubiquitylated GLT1 accumulates in the intracellular compartment. However, internalization of ubiquitylated GLT1 was blocked with a dominant negative dynamin 2 mutant, indicating that the addition of ubiquitin moieties to the transporter in the membrane precedes its endocytosis. The elimination of lysines from the C-terminus of the transporter (lysines 497, 517, 526, 550, 558, 570, and 573) blocked GLT1 ubiquitylation and endocytosis. However, reintroduction of lysine 517 alone into this mutant was sufficient to restore PMA dependent ubiquitylation and internalization of GLT1. Similarly, reintroduction of lysine 526 restored the endocytosis, while this was only partially recovered after the individual reintroduction of lysines 550 or 570. These data suggest that the activation of PKC induces the ubiquitylation of these C-terminal lysine residues in GLT1 and that this modification mediates the interaction of the transporter with the endocytic machinery.

The glutamate transporter GLT1b interacts with the scaffold protein PSD-95.

The glutamate transporter (GLT1) regulates glutamate concentrations in glutamatergic synapses and it is expressed in at least two isoforms, GLT1a and GLT1b. In this work, we show that the C-terminus of GLT1b is able to interact with the PDZ domains of a number of proteins. Notably, one of them might be the scaffold protein post-synaptic density (PSD-95). GLT1b formed co-immunoprecipitable complexes with PSD-95 in solubilizated rat brain extracts, complexes that also contained NMDA receptors. Co-transfection of GLT1b, PSD-95, and NMDA receptor subunits in heterologous expression systems recapitulated in vitro the interactions among these proteins that had been observed in the rat brain extracts and revealed the importance of the GLT1b C-terminal PDZ binding motif in tethering this transporter to PSD-95. Significantly, co-expression of GLT1b and PSD-95 increased the V(max) of the transporter by decreasing the rate of GLT1b endocytosis. Moreover, GLT1b transfected into primary cultured neurons or glia formed protein clusters that co-localized with co-transfected PSD-95, clusters that in these neurons accumulated preferentially in dendritic spines. We hypothesize that the GLT1b/PSD-95 interaction, characterized here in vitro, might anchor this transporter close to the post-synaptic glutamate receptors, thereby permitting the fine regulation of glutamate concentrations in this microenvironment. This tight association might also facilitate the regulation of GLT1b through the signaling pathways initiated by the activation of glutamate receptors.