Repurposed drugs block toxin-driven platelet clearance by the hepatic Ashwell-Morell receptor to clear Staphylococcus aureus bacteremia

Staphylococcus aureus (SA) bloodstream infections cause high morbidity and mortality (20 to 30%) despite modern supportive care. In a human bacteremia cohort, we found that development of thrombocytopenia was correlated to increased mortality and increased α-toxin expression by the pathogen. Platelet-derived antibacterial peptides are important in bloodstream defense against SA, but α-toxin decreased platelet viability, induced platelet sialidase to cause desialylation of platelet glycoproteins, and accelerated platelet clearance by the hepatic Ashwell-Morell receptor (AMR). Ticagrelor (Brilinta), a commonly prescribed P2Y12 receptor inhibitor used after myocardial infarction, blocked α-toxin–mediated platelet injury and resulting thrombocytopenia, thereby providing protection from lethal SA infection in a murine intravenous challenge model. Genetic deletion or pharmacological inhibition of AMR stabilized platelet counts and enhanced resistance to SA infection, and the anti-influenza sialidase inhibitor oseltamivir (Tamiflu) provided similar therapeutic benefit. Thus, a “toxin-platelet-AMR” regulatory pathway plays a critical role in the pathogenesis of SA bloodstream infection, and its elucidation provides proof of concept for repurposing two commonly prescribed drugs as adjunctive therapies to improve patient outcomes.

A CD209 ligand and a sialidase inhibitor differentially modulate adipose tissue and liver macrophage populations and steatosis in mice on the Methionine and Choline-Deficient (MCD) diet

Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and type 2 diabetes and is characterized by the accumulation of fat in the liver (steatosis). NAFLD can transition into non-alcoholic steatohepatitis (NASH), with liver cell injury, inflammation, and an increased risk of fibrosis. We previously found that injections of either 1866, a synthetic ligand for the lectin receptor CD209, or DANA, a sialidase inhibitor, can inhibit inflammation and fibrosis in multiple animal models. The methionine and choline-deficient (MCD) diet is a model of NASH which results in the rapid induction of liver steatosis and inflammation. In this report, we show that for C57BL/6 mice on a MCD diet, injections of both 1866 and DANA reversed MCD diet-induced decreases in white fat, decreases in adipocyte size, and white fat inflammation. However, these effects were not observed in type 2 diabetic db/db mice on a MCD diet. In db/db mice on a MCD diet, 1866 decreased liver steatosis, but these effects were not observed in C57BL/6 mice. There was no correlation between the ability of 1866 or DANA to affect steatosis and the effects of these compounds on the density of liver macrophage cells expressing CLEC4F, CD64, F4/80, or Mac2. Together these results indicate that 1866 and DANA modulate adipocyte size and adipose tissue macrophage populations, that 1866 could be useful for modulating steatosis, and that changes in the local density of 4 different liver macrophages cell types do not correlate with effects on liver steatosis.

Repurposed Drugs Block Toxin-Driven Platelet Clearance by the Hepatic Ashwell-Morell Receptor to Clear Staphylococcus aureus Bacteremia

ABSTRACTStaphylococcus aureus (SA) bloodstream infections cause high morbidity and mortality (20-30%) despite modern supportive care. In a human bacteremia cohort, development of thrombocytopenia was correlated to increased mortality and increased α-toxin expression by the pathogen. Platelet-derived antibacterial peptides are important in bloodstream defense against SA, but α-toxin decreased platelet viability, induced platelet sialidase to cause desialylation of platelet glycoproteins, and accelerated platelet clearance by the hepatic Ashwell-Morell receptor (AMR). Ticagrelor (Brilinta®), a commonly prescribed P2Y12 receptor inhibitor used post-myocardial infarction, blocked α-toxin-mediated platelet injury and resulting thrombocytopenia, thus providing protection from lethal SA infection in a murine intravenous challenge model. Genetic deletion or pharmacological inhibition of AMR stabilized platelet counts and enhanced resistance to SA infection, and the anti-influenza sialidase inhibitor oseltamivir (Tamiflu®) provided similar therapeutic benefit. Thus a “toxin-platelet-AMR” regulatory pathway plays a critical role in the pathogenesis of SA bloodstream infection, and its elucidation provides proof-of-concept for repurposing two FDA-approved drugs as adjunctive therapies to improve patient outcomes.

Autoantibody Mediated Desialylation Impairs Human Thrombopoiesis and Platelet Life Span

Background: The low platelet count in autoimmune thrombocytopenia (ITP) is caused by enhanced destruction of opsonised platelets in the spleen upon binding of the anti-platelet autoantibodies (AAbs) to the glycoproteins (GPs) express on PLT's surface. Data from animal model suggested that desialylation may contribute to PLT destruction in ITP. However, accumulating evidence suggests that reduction of PLT generation from megakaryocytes (MKs) in bone morrow is also responsible thrombocytopenia in ITP. Based on these considerations, we hypothesized that AAb-mediated desialylation of the GPs expressed on PLT and MKs may interfere with PLT formation and life span. Methods: Sera from 100 ITP patients were investigated in this study. AAb-induced desialylation was detected using a lectin binding assay (LBA) by flow cytometry (FC). To investigate the impact of desialylation on the life-span of human PLTs, the NSG mouse model was used. PLTs and MKs functions were assessed after AAb treatment using proplatelet formation test and adhesion assays on different surfaces. Results: Sera from 35/100 (35%) ITP patients induced cleavage of sialic acid from PLT surface. Injection of desialylating AAbs in vivo resulted in accelerated clearance of human PLTs which was significantly reduced by a specific sialidase inhibitor that prevents desialylation on the PLT surface (survival after 5h: 29%, range 22-40% vs. 48%, range 41-53%, p=0.014, respectively). Desialylating AAbs caused a significant reduction in PLT adhesion to fibrinogen and von Willebrand factor (mean of % adherent PLTs compared to control IgG: 34±6%, p=0.004 and 26±2%, p=0.001, respectively). Interestingly, PLT adhesion was recovered in the presence of a sialidase inhibitor (mean of % adherent PLTs: 86±6%, p=0.001 and 67±10, p=0.020, respectively). IgG fractions from 7/10 (70%) ITP-sera were able to cleave sialic acid and induce exposure of ß-galactose residues on CD34+-derived MKs. Desialylating AAbs induced lower ability to form proplatelet extensions compared to control IgG, which was significantly increased in the presence of the sialidase inhibitor (mean of % proplatelet forming MKs: 42±11% vs. 90±9%, p=0.032, respectively). Conclusion: Our findings show that AAbs from a subgroup of ITP patients are not only able to cleave sialic acid on surface of human PLTs, but also on MKs leading to accelerate PLT destruction and impaired thrombopoiesis, respectively. In addition, we observed that AAb-mediated receptor desialyation interferes with cell interaction with extracellular matrix proteins leading to impaired PLT adhesion, MK differentiation and thrombopoiesis. These novel findings highlight the multiple effects of AAbs in ITP and add to the existing evidence that ITP is rather a group of disorders sharing common characteristics, namely loss of immune tolerance toward PLT and MK antigens and increased bleeding tendency. Disclosures No relevant conflicts of interest to declare.

Desialylation by Edwardsiella tarda is the initial step in the regulation of its invasiveness

Edwardsiella tarda is a gram-negative bacterium causing significant economic losses to aquaculture. E. tarda possesses NanA sialidase which removes sialic acids from α2–3 sialo-glycoprotein of host cells. However, the relationship between NanA sialidase activity and E. tarda invasiveness remains poorly understood. Furthermore, the pathway of sialic acid metabolism in E. tarda remains to be elucidated. We studied sialidase activity in several E. tarda strains and found that the pathogenic strains exhibited higher sialidase activity and greater up-regulation of the NanA mRNA level than non-pathogenic strain. Pathogenic strains also showed higher rates of infection in GAKS cells, and the infection was drastically suppressed by sialidase inhibitor. Additionally, NanA gene overexpression significantly increased infection and treatment of E. tarda with free sialic acid enhanced the rate of infection in GAKS cells. Sialic acid treatment enhanced mRNA levels of two N-acetylneuraminate lyases and one N-acetylneuraminate cytidylyltransferase. E. tarda uses sialic acid as a carbon source for growth via N-acetylneuraminate lyases. The strains with high N-acetylneuraminate cytidylyltransferase level showed greater sialylation of the lipopolysaccharides and glycoproteins. Our study establishes the significance of desialylation by E. tarda sialidase in the regulation of its invasiveness.

Desialylation of Megakaryocytes Diminishes Platelet Production By Disrupting Megakaryocyte Adhesion, Migration and Proplatelet Formation in Chronic Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a common autoimmune disorder characterized by increased bleeding tendency and isolated thrombocytopenia. The precise pathogenesis of the decreased thrombopoiesis in chronic ITP (cITP) is poorly understood. Megakaryocytes (MKs) in cITP show impaired maturation and thrombopoiesis, which are correlated with numerous glycoproteins on the surface of MKs. Different types of sialoglycoproteins are expressed on the surface of megakaryocytes, including GPIbα and platelet endothelial cell adhesion molecule-1 (PECAM-1), both of which participate in megakaryocyte migration to the vascular niche in the bone marrow (BM) and in proplatelet formation. Desialylation has recently been identified a contributor to the pathogenesis of thrombocytopenia. Our previous study has demonstrated that desialylation of GPIbα is related to increased apoptosis and phagocytosis of platelets in cases of prolonged isolated thrombocytopenia after allogeneic hematopoietic stem cell transplantation (Zhang et al., J Hematol Oncol, 2015). Because MKs are heavily sialylated cells, we raised the question whether the desialylation of megakaryocytes contributes to the defective thrombopoiesis in patients with cITP through impaired MK migration, adhesion and proplatelet formation in the vascular niche. MK desialylation was analyzed by flow cytometry using lectins. Desialylated glycoproteins were measured using selective exo-enzymatic labeling. Protein expression, distribution and interaction were measured using the following techniques: immunofluorescence, flow cytometry, western blot and immunoprecipitation. cITP MKs exhibited increased β-galactose exposure compared to the control MKs, indicating excessive desialylation. Desialylation was correlated with decreased platelet production of MKs. We further explored the cause of desialylation and found that the sialidase NEU1 was over-expressed in MKs. Treatment with the sialidase inhibitor DANA ameliorated the loss of sialic acids. These results indicated that NEU1 contributed to the desialylation of MKs in cITP. Altered MK distribution in the BM niche was exhibited upon BM biopsy of cITP patients. The ratio of perivascular MKs was markedly decreased in cITP patients. Defective adhesion and transmigration behaviors were also discovered in desialylated cITP MKs. The motility of cITP MKs through stromal cell monolayers driven by stromal cell derived factor 1 (SDF1) was decreased. Adherence to fibronectin, collagen and fibrinogen was assessed, and desialylated cITP MKs exhibited an increase in adhesion with these macromolecules. Similar abnormalities were observed in the BM niche of ST6Gal1-/- mice, and treatment with ST6Gal1 and CMP-SA augmented the ratio of MKs in the BM vascular niche in ST6Gal1-/-mice, indicating that desialylation impaired the MK migration and adhesion. Additional experiments focused on which specific sialoglycoproteins are excessively desialylated. As detected by SEEL, PECAM-1 exhibited excessive desialylation in cITP MKs, which was related to impaired CXCR4 polarization in response to SDF1. Inhibition of sialidase using DANA partially restored this polarization, demonstrating that desialylation of PECAM-1 was responsible for the defect in MK migration to the vascular niche. Meanwhile, PECAM-1 desialylation was associated with GPIIb/IIIa overactivation, which correlated to the increased adhesion of MKs. This increased adhesion was reversed by the GPIIb/IIIa inhibitor lotrafiban, indicating that desialylated PECAM-1 contributed to the abnormal adhesion via overactivation of GPIIb/IIIa. Desialylation of GPIbα was found on the surface of MKs from cITP patients and was associated with abnormal microtubule formation and increased MK apoptosis through altered 14-3-3ζ distribution, which led to the impediment of proplatelet formation in the vascular niche. In conclusion, our results demonstrate that MKs are desialylated by NEU1 in cITP patients, and desialylated MKs present with defective migration towards the vascular niche, abnormal adhesion to the extracellular matrix and impaired proplatelet formation. Desialylation of PECAM-1 and GPIbα have been demonstrated to be responsible for these abnormal behaviors of MKs. Sialidase inhibitor shows an improvement in the thrombopoiesis of cITP MKs; therefore, our study implies a novel potential approach for the treatment of cITP. Disclosures No relevant conflicts of interest to declare.