Mehrab Nasirikenari

Fluorinated per-acetylated GalNAc metabolically alters glycan structures on leukocyte PSGL-1 and reduces cell binding to selectins

Novel strategies to control the binding of adhesion molecules belonging to the selectin family are required for the treatment of inflammatory diseases. We tested the possibility that synthetic monosaccharide analogs can compete with naturally occurring sugars to alter the O-glycan content on human leukocyte cell surface selectin-ligand, P-selectin glycoprotein ligand-1 (PSGL-1). Resulting reduction in the sialyl Lewis-X-bearing epitopes on this ligand may reduce cell adhesion. Consistent with this hypothesis, 50muM per-acetylated 4F-GalNAc added to the growth media of promyelocytic HL-60 cells reduced the expression of the cutaneous lymphocyte associated-antigen (HECA-452 epitope) by 82% within 2 cell doubling cycles. Cell binding to all 3 selectins (L-, E-, and P-selectin) was reduced in vitro. 4F-GalNAc was metabolically incorporated into PSGL-1, and this was accompanied by an approximately 20% reduction in PSGL-1 glycan content. A 70% to 85% reduction in HECA-452 binding epitope and N-acetyl lactosamine content in PSGL-1 was also noted on 4F-GalNAc addition. Intravenous 4F-GalNAc infusion reduced leukocyte migration to the peritoneum in a murine model of thioglycolate-induced peritonitis. Thus, the compound has pharmacologic activity. Overall, the data suggest that 4F-GalNAc may be applied as a metabolic inhibitor to reduce O-linked glycosylation, sialyl Lewis-X formation, and leukocyte adhesion via the selectins.

Anti-inflammatory IgG Production Requires Functional P1 Promoter in β-Galactoside α2,6-Sialyltransferase 1 (ST6Gal-1) Gene

Background: β-Galactoside α2,6-sialyltransferase 1 (ST6Gal-1) action is essential for the anti-inflammatory activity in intravenous immunoglobulin (IVIG) therapy. Results: Fc sialylation changes in accordance to the severity of inflammation. Inactivation of the P1 promoter abrogated IgG Fc sialylation. Conclusion: Fc sialylation depends on ST6Gal-1 in the circulation. Defective Fc sialylation is a mechanism for the generally proinflammatory tendencies of the P1-ablated mutant mouse (Siat1ΔP1). Significance: Anti-inflammatory bioactivity of IVIG requires sialylated Fc. The anti-inflammatory properties associated with intravenous immunoglobulin therapy require the sialic acid modification of the N-glycan of the Fc domain of IgG. Sialylation of the Fc fragment is mediated by β-galactoside α2,6-sialyltransferase 1 (ST6Gal-1), acting on the Gal(β4)GlcNAc terminal structure of the biantennary N-glycans on the Fc domain. However, little is known regarding the in vivo regulation of Fc sialylation and its role in the progression of inflammatory processes. Here, we report that decreased Fc sialylation of circulatory IgG accompanies the acute phase response elicited by turpentine exposure or upon acute exposure to either nontypeable Haemophilus influenzae or ovalbumin. However, Fc sialylation was increased 3-fold from the base line upon transition to chronic inflammation by repeated exposure to challenge. The P1 promoter of the ST6Gal-1 gene is critical for Fc sialylation, but P1 does not drive ST6Gal-1 expression in B cells. The Siat1ΔP1 mouse, with a dysfunctional P1 promoter, was unable to produce sialylated Fc in the systemic circulation, despite the presence of Gal(β4)GlcNAc termini on the Fc glycans. The major contribution of P1 action is to synthesize ST6Gal-1 enzymes that are deposited into the systemic circulation. The data strongly indicate that this pool of extracellular ST6Gal-1 in the blood impacts the sialylation of IgG Fc and that defective Fc sialylation is likely a major contributing mechanism for the proinflammatory tendencies previously noted in Siat1ΔP1 animals.

Role for Hepatic and Circulatory ST6Gal-1 Sialyltransferase in Regulating Myelopoiesis

Recent findings have established a role for the ST6Gal-1 sialyltransferase in modulating inflammatory cell production during Th1 and Th2 responses. ST6Gal-1 synthesizes the Sia(α2,6) to Gal(β1,4)GlcNAc linkage on glycoproteins on cell surfaces and in systemic circulation. Engagement of P1, one of six promoter/regulatory regions driving murine ST6Gal-1 gene expression, generates the ST6Gal-1 for myelopoietic regulation. P1 utilization, however, is restricted to the liver and silent in hematopoietic cells. We considered the possibility that myelopoiesis is responsive to the sialylation of liver-derived circulatory glycoproteins, such that reduced α2,6-sialylation results in elevated myelopoiesis. However, 2-dimensional differential in gel electrophoresis (2D-DIGE) analysis disclosed only minimal alterations in the sialylation of sera glycoproteins of ST6Gal-1-deficient mice when compared with wild-type controls, either at baseline or during an acute phase response when the demand for sialylation is greatest. Furthermore, sera from ST6Gal-1-deficient animals did not enhance myelopoietic activity in ex vivo colony formation assays. Whereas there was only minimal consequence to the α2,6-sialylation of circulatory glycoproteins, ablation of the P1 promoter did result in strikingly depressed levels of ST6Gal-1 released into systemic circulation. Therefore, we considered the alternative possibility that myelopoiesis may be regulated not by the hepatic sialyl glycoproteins, but by the ST6Gal-1 that was released directly into circulation. Supporting this, ex vivo colony formation was notably attenuated upon introduction of physiologic levels of ST6Gal-1 into the culture medium. Our data support the idea that circulatory ST6Gal-1, mostly of hepatic origin, limits myelopoiesis by a mechanism independent of hepatic sialylation of serum glycoproteins.

Remodeling of Marrow Hematopoietic Stem and Progenitor Cells by Non-self ST6Gal-1 Sialyltransferase

Background: The circulatory ST6Gal-1 level is inversely related to hematopoietic activity, but the biochemical function of systemic ST6Gal-1 is unknown. Results: Hematopoietic progenitors do not express self-ST6Gal-1 but are acted upon by remotely produced enzyme. Conclusion: Distally produced rather than endogenous ST6Gal-1 is the principal modifier of the early hematopoietic progenitor cell surface. Significance: Extrinsic ST6Gal-1 may be a potent systemic regulator of hematopoiesis. Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin−c-Kit+ and Lin−Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin−Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.

Platelets support extracellular sialylation by supplying the sugar donor substrate

Background: ST6Gal-1 sialyltransferase is a prominent circulatory glycosyltransferase whose function remained in question because an extracellular source of sugar donor substrate was unknown. Results: Extracellular ST6Gal-1 catalysis uses platelet-supplied sugar donors. Conclusion: Activated platelets release donor substrates to functionally drive extracellular glycosylation by soluble ST6Gal-1. Significance: Remodeling by extracellular glycosyltransferases may be an important mechanism to generate physiologically important glycans. Sizable pools of freely circulating glycosyltransferases are in blood, but understanding their physiologic contributions has been hampered because functional sources of sugar donor substrates needed to drive extracellular glycosylation have not been identified. The blood-borne ST6Gal-1 produced and secreted by the liver is the most noted among the circulatory glycosyltransferases, and decorates marrow hematopoietic progenitor cells with α2,6-linked sialic acids and restricts blood cell production. Platelets, upon activation, secrete a plethora of bioactive molecules including pro- and anti-inflammatory mediators. Cargos of sugar donor substrates for glycosyltransferase activity have also been reported in platelets. Here, we implemented a cell-based system to interrogate platelets for their ability to deliver effectively the sugar donor substrate for extracellular ST6Gal-1 to function. We report that thrombin-activated platelets, at physiologic concentration and pH, can efficiently and effectively substitute for CMP-sialic acid in extracellular ST6Gal-1-mediated sialylation of target cell surfaces. Activated platelets can also supply the sialic acid donor to sialylate the synthetic acceptor, Gal(β1,4)GlcNAcα-o-benzyl, with the product Sia(α2,6)Gal(β1,4)GlcNAcα-o-benzyl structurally confirmed by LC/MS. Platelet-secreted donor substrate was recovered in the 100,000 × g sediment, strongly suggesting the association of this otherwise soluble substrate, putatively CMP-sialic acid, within platelet microparticles. Sequestration within microparticles may facilitate delivery of glycosylation substrate at effective dosages to sites of extracellular glycosylation while minimizing excessive dilution.

Altered eosinophil profile in mice with ST6Gal-1 deficiency: an additional role for ST6Gal-1 generated by the P1 promoter in regulating allergic inflammation

Cumulative evidence indicates that the sialyltransferase ST6Gal‐1 and the sialyl‐glycans, which it constructs, are functionally pleiotropic. Expression of the ST6Gal‐1 gene is mediated by six distinct promoter/regulatory regions, and we hypothesized that these promoters may be used differentially to produce ST6Gal‐1 for different biologic purposes. To examine this hypothesis, we compared a mouse with a complete deficiency in ST6Gal‐1 (Siat1 null) with another mouse that we have created previously with a disruption only in the P1 promoter (Siat1ΔP1). We noted previously greater neutrophilic inflammation associated with ST6Gal‐1 deficiency. Here, we report that ST6Gal‐1‐deficient mice also have significantly elevated eosinophilic responses. Upon i.p. thioglycollate elicitation, eosinophils accounted for over 20% of the total peritoneal inflammatory cell pool in ST6Gal‐1‐deficient animals, which was threefold greater than in corresponding wild‐type animals. A principal feature of allergic respiratory inflammation is pulmonary eosinophilia, we evaluated the role of ST6Gal‐1 in allergic lung inflammation. Using OVA and ABPA experimental models of allergic airways, we showed that ST6Gal‐1 deficiency led to greater airway inflammation characterized by excessive airway eosinophilia. The severity of airway inflammation was similar between Siat1ΔP1 and Siat1 null mice, indicating a role for P1‐generated ST6Gal‐1 in regulating eosinophilic inflammation. Colony‐forming assays suggested greater IL‐5‐dependent eosinophil progenitor numbers in the marrow of ST6Gal‐1‐deficient animals. Moreover, allergen provocation of wild‐type mice led to a significant reduction in P1‐mediated ST6Gal‐1 mRNA and accompanied decline in circulatory ST6Gal‐1 levels. Taken together, the data implicate ST6Gal‐1 as a participant in regulating not only Th1 but also Th2 responses, and ST6Gal‐1 deficiency can lead to the development of more severe allergic inflammation with excessive eosinophil production.

Nrf2 Regulates Chronic Lung Inflammation and B-Cell Responses to Nontypeable Haemophilus influenzae

Nrf2 is a leucine zipper transcription factor that protects against oxidant-induced injury. Nontypeable Haemophilus influenzae is responsible for frequent disease exacerbations in patients with chronic obstructive pulmonary disease and is responsible for causing otitis media in young children. We hypothesized that Nrf2 would limit inflammatory responses to nontypeable H. influenzae. The objective of this study was to assess the role of Nrf2 in chronic lung inflammation and regulation of immune responses to nontypeable H. influenzae in mice. Wild-type (C57BL/6) mice and Nrf2(-/-) mice were instilled by oropharyngeal aspiration of 1 × 10(6) colony-forming units of live, nontypeable H. influenzae (NTHI) twice a week for 4 to 16 consecutive weeks to generate a chronic inflammatory milieu within the lungs that models chronic bronchitis. Nrf2(-/-) mice had increased lymphocytic airway inflammation compared with WT mice after NTHI challenge. Although the extent of NTHI-induced peribronchovascular inflammation did not significantly differ between the genotypes, plasma cell infiltration was significantly more abundant in Nrf2(-/-) mice. Most strikingly, Nrf2(-/-) mice generated significantly enhanced and persistent levels of serum antibodies against P6, a key outer membrane protein of NTHI. Lung dendritic cells from Nrf2(-/-) mice challenged with NTHI had increased activation markers compared with dendritic cells from similarly treated WT mice. Nrf2 regulates NTHI-induced airway inflammation characterized by lymphocytic and plasma cell infiltration and the activation of lung dendritic cells and B-cell responses in mice. Nrf2 may be a potential therapeutic target in limiting the bacterial infection-induced airway inflammation that drives exacerbations of chronic obstructive pulmonary disease.

The blood-borne sialyltransferase ST6Gal-1 is a negative systemic regulator of granulopoiesis

Responding to systemic demands in producing and replenishing end‐effector blood cells is predicated on the appropriate delivery and interpretation of extrinsic signals to the HSPCs. The data presented herein implicate the systemic, extracellular form of the glycosyltransferase ST6Gal‐1 in the regulation of late‐stage neutrophil development. ST6Gal‐1 is typically a membrane‐bound enzyme sequestered within the intracellular secretory apparatus, but an extracellular form is released into the blood from the liver. Both human and murine HSPCs, upon exposure to extracellular ST6Gal‐1 ex vivo, exhibited decreased proliferation, diminished expression of the neutrophilic primary granule protein MPO, and decreased appearance of CD11b+ cells. HSPC suppression was preceded by decreased STAT‐3 phosphorylation and diminished C/EBPα expression, without increased apoptosis, indicating attenuated G‐CSF receptor signaling. A murine model to raise systemic ST6Gal‐1 level was developed to examine the role of the circulatory enzyme in vivo. Our results show that systemic ST6Gal‐1 modified the cell surface of the GMP subset of HSPCs and decreased marrow neutrophil reserves. Acute airway neutrophilic inflammation by LPS challenge was used to drive demand for new neutrophil production. Reduced neutrophil infiltration into the airway was observed in mice with elevated circulatory ST6Gal‐1 levels. The blunted transition of GMPs into GPs in vitro is consistent with ST6Gal‐1‐attenuated granulopoiesis. The data confirm that circulatory ST6Gal‐1 is a negative systemic regulator of granulopoiesis and moreover suggest a clinical potential to limit the number of inflammatory cells by manipulating blood ST6Gal‐1 levels.

Leukocyte-borne α(1,3)-fucose is a negative regulator of β2-integrin-dependent recruitment in lung inflammation

Leukocyte recruitment in inflammation is a multistep, sequential cascade where the initial step is the selectin‐dependent tethering, followed by the formation of firmer integrin‐mediated adhesive forces leading to extravasation. The α(1,3)‐fucose‐containing sialyl‐Lewis X (sLeX) is the archetypical ligand on leukocyte surfaces mediating selectin interactions. Canonically, disruption of α(1,3)‐fucose formation ablates selectin‐mediated adhesion, dramatically reducing trafficking. We report a paradoxical response to α(1,3)‐fucose deficiency in which the loss exacerbated rather than attenuated leukocyte recruitment in a murine model of acute airway inflammation. The architecture of the capillary‐dominated vasculature in the lung minimized the importance of the selectin dependent step, and we observed that α(1,3)‐fucose deficiency augmented CXCR2‐mediated Rap1‐GTP signaling to enhance the β2‐integrin‐ICAM‐1‐binding axis. The data disclose a previously unknown function for α(1,3)‐fucose, in which this structure negatively regulates the integrin activation step in leukocyte recruitment.