Recent reports have documented that extracellular sialyltransferases can remodel both cell-surface and secreted glycans by a process other than the canonical cell-autonomous glycosylation that occurs within the intracellular secretory apparatus. Despite association of the abundance of these extracellular sialyltransferases, particularly ST6Gal-1, with disease states such as cancer and a variety of inflammatory conditions, the prevalence of this extrinsic glycosylation pathway remains unknown. Here we observed no significant extrinsic sialylation in resting mice, suggesting that extrinsic sialylation is not a constitutive process. However, extrinsic sialylation in the periphery could be triggered by inflammatory challenges, such as exposure to ionizing radiation or to bacterial lipopolysaccharides. Sialic acids from circulating platelets were used to remodel target cell surfaces. Platelet activation was minimally sufficient to elicit extrinsic sialylation, as demonstrated with the FeCl model of mesenteric artery thrombosis. Although extracellular ST6Gal-1 supports extrinsic sialylation, other sialyltransferases are present in systemic circulation. We also observed extrinsic sialylation in animals deficient in ST6Gal-1, demonstrating that extrinsic sialylation is not mediated exclusively by ST6Gal-1. Together, these observations form an emerging picture of glycans biosynthesized by the canonical cell-autonomous glycosylation pathway, but subjected to remodeling by extracellular glycan-modifying enzymes.
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.
The production of new blood cells relies on a hierarchical network of hematopoietic stem and progenitor cells (HSPCs). To maintain lifelong hematopoiesis, HSPCs must be protected from ionizing radiation or other cytotoxic agents. For many years, murine models have been a valuable source of information regarding factors that either enhance or reduce the survival of HSPCs after exposure of marrow to ionizing radiation. In a recent series of studies, however, it has become clear that housing-related factors such as the cool room temperature required for laboratory mice can exert a surprising influence on the outcome of experiments. Here we report that the mild, but chronic cold-stress endured by mice housed under these conditions exerts a protective effect on HSPCs after both non-lethal and lethal doses of total body irradiation (TBI). Alleviation of this cold-stress by housing mice at a thermoneutral temperature (30°C) resulted in significantly greater baseline radiosensitivity to a lethal dose of TBI with more HSPCs from mice housed at thermoneutral temperature undergoing apoptosis following non-lethal TBI. Cold-stressed mice have elevated levels of norepinephrine, a key molecule of the sympathetic nervous system that binds to β-adrenergic receptors. We show that blocking this signaling pathway in vivo through use of the β-blocker propanolol completely mitigates the protective effect of cold-stress on HSPC apoptosis. Collectively this study demonstrates that chronic stress endured by the standard housing conditions of laboratory mice increases the resistance of HSPCs to TBI-induced apoptosis through a mechanism that depends upon β-adrenergic signaling. Since β-blockers are commonly prescribed to a wide variety of patients, this information could be important when predicting the clinical impact of HSPC sensitivity to TBI.
The dynamic interaction of hematopoietic stem and progenitor cells (HSPC) with niche environments and delivery of soluble extrinsic signals are the principal driving factors determining cell fate decisions for self renewal, expansion, and differentiation. Here we present evidence that a systemic glycosyltransferase originating from the liver is one such extrinsic signal that acts by remodeling hematopoietic progenitor cell surfaces. There is mounting evidence that sialyl- and fucosyl-glycan structures mediate adhesive interactions critical to stem cell differentiation (Alisson-Silva et al. Glycobiology, 2014). Our lab has discovered that the absence of a single type of sialic acid linkage on HSPC cell surface profoundly augments the ability of hematopoietic progenitors to produce myeloid cells. This single sialic acid linkage, the Sia(alpha2,6) linkage on Gal(beta1,4)-GlcNAc termini, is constructed by the blood-borne glycosyltransferase ST6Gal-1. Mice with diminished circulatory ST6Gal-1 levels have more severe inflammatory responses, secondary to elevated production of granulocytic inflammatory cells in the marrow. Less clear, however, are the consequences of elevated systemic ST6Gal-1 on HSPC homeostasis. Here, we show that ST6Gal-1 acts as a cell-signaling rheostat by blunting the response of granulocytic progenitors to cytokines. Accordingly, ex vivo ST6Gal-1 treated human CD34+ cells showed stunted growth response to a myeloid-differentiation cocktail consisting of SCF, IL-3, and G-CSF (Fig. 1). Blinded cytochemical myeloperoxidase evaluation noted a substantial decrease in myeloid differentiation in ST6Gal-1 treated cells. To study these effects in vivo, we utilized a transplantable murine melanoma (B16-S6G) transduced to stably express excess soluble ST6Gal-1 into circulation in mice. We subjected naïve B16-S6G, B16-cntl, and C57BL/6 (WT) animals to a Th1 inflammatory challenge with intra-tracheal lipopolysaccharide (LPS) and monitored inflammatory cell infiltrates in the bronchial alveolar lavage fluid (BALF). The mutant dP1 mouse, which is deficient in the circulatory pool of extracellular ST6Gal-1, was included as positive control for enhanced inflammation (Nasirikenari et al. Journal of Leukocyte Biology, 2010). On average, around 1.5x106 neutrophils were recoverable in the bronchial alveolar lavage fluid in B16-S6G mice 24 hours after LPS challenge. This represents about half the recovered cells from B16-cntl and WT mice, and over a 3-fold reduction from dP1 animals. Although total cellularity in the marrow was not perturbed amongst the different models, we found the marrow to be drastically depleted (>50% of normal) of CD11b+Ly6G+ PMNs. Higher order marrow progenitors within the Lin-, c-kit+, Sca-1- (LK) population, namely common myeloid progenitor (CMP; LK, CD41-), pre-granulocyte macrophage progenitor (Pre-GM; LK, CD41-FcgRII/III-Endoglin-CD150-), and pro-granulocyte macrophage progenitor (pro-GMP; LK, CD41-FcgRII/III+) numbers remained similar across control and transduced tumor-bearing mice. However, enhanced surface sialylation segregated markedly with granulocytic precursors, and ex vivo primary marrow culture revealed ST6Gal-1 blunted granulocytic differentiation at the level of the granulocyte-macrophage progenitor (GMP). Taken together, our data show that ST6Gal-1 action conveys instructive cues critical for late-stage granulocytic development. These findings have important implications for instances in which suppression of an overactive innate response may be clinically desirable. Disclosures No relevant conflicts of interest to declare.
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