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

Abstract

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.

Keywords: glycosylation; hematopoiesis; neutrophils; sialylation.

Figure 1.. Neutrophil lung infiltration and inflammation correlates inversely with circulatory ST6Gal-1.

WT, dP1, B16-cntl, and B16-S6G mice were subjected to 50 μg of LPS by oropharyngeal instillation. At 18 h, the BALF was collected and the neutrophils counted. Serum samples were collected for enzyme analysis. (A) Sialyltransferase activities in the sera were measured by following the transfer of CMP-[3H]Sia to Galβ1–4GlcNAc-O-Bn (LacNAc). The α2,6-Sia product formed by ST6Gal-1 is shown (n = 3). ***P < 0.001. (B) Total number of neutrophils recovered from the BALF of LPS-induced animals (n = 3). *P < 0.05. (C and D) B16-cntl (C) and B16-S6G (D) show a decreased number of infiltrated neutrophils at 18 h. Arrows: infiltrating neutrophils around the bronchiole and within the bronchiolar epithelium.

Figure 2.. Depleted marrow neutrophil reservoir in B16-S6G mice.

(A) Total marrow cellularity per femur. (B) Total cells per femur found to express PMN surface phenotype (CD11b⁺Ly6G⁺) vs. non-PMNs (CD11⁻Ly6G⁻) (n = 3). ***P < 0.001. (C) Representative flow cytometry plots depicting the population shift quantified in (B).

Figure 3.. ST6Gal-1 modulates surface α2,6 sialylation levels on GPs in vivo.

WT, B16-cntl, and B16-S6G marrow was isolated, and both the α2,6 surface sialylation and the specific bone marrow subsets were analyzed by flow cytometry. (A) Lineage commitment of progenitors upstream of granulopoiesis. (B) SNA profiles of total bone marrow, CMPs, Pre-GMs, FcγRII/III⁺ progenitors, and mature granulocytes. (C) Quantification of SNA staining on cells expressing FcγRII/III⁺ progenitor markers and distribution by CD115 (M-CSFR) expression (n = 4). *P < 0.05; **P < 0.01. (D) WT marrow from the GMP, GP, and MP subsets were assessed for SNA staining.

Figure 4.. ST6Gal-1 suppresses neutrophilic, but not monocytic, differentiation of GMPs.

FcγRII/III⁺ progenitors (0.75 × 10⁵) enriched from pooled WT marrow were expanded in culture in the presence of murine G-CSF, SCF, and IL-3. ST6Gal-1-treated cells were subjected to 3 h of ex vivo sialylation before expansion. At days 4 and 6, samples were counted and analyzed by flow cytometry for lineage markers. Cells undergoing apoptosis were enumerated on d 7, with annexin V and PI staining. In a separate experiment, GMPs enriched from pooled WT marrow were expanded in culture in the presence of murine G-CSF, M-CSF, SCF, and IL-3. After 3 d, samples were analyzed by flow cytometry for lineage markers. (A) Total cells per well recovered after 4 and 6 d in culture. Dashed line: day 0 cell counts (n = 4). *P < 0.05. (B) Quantification of total CD11b⁺ Gr-1⁺ neutrophils per well recovered after 6 d in culture (n = 4). **P < 0.01. (C) Apoptotic index of treated vs. untreated cells. Early apoptotic cells were defined as annexin V^hiPI^lo; late apoptotic cells were defined as annexin V^hiPI^hi. NS, not significant. (D) Representative flow plots showing GMP neutrophil differentiation. Gate indicates CD11b⁺ Ly6C low Ly6G⁺ neutrophils. Percentages are of total cells recovered. (E) Quantification of total CD11b⁺ Ly6C low Ly6G⁺ neutrophils and CD11b⁺ Ly6C⁺ monocytes per well (n = 5). **P < 0.01.

Figure 5.. ST6Gal-1 inhibits G-CSF signaling and GP formation.

LK cells enriched from pooled WT marrow were expanded in culture in the presence of murine G-CSF, SCF, and IL-3. ST6Gal-1-treated cells were subjected to 3 h of ex vivo sialylation before expansion. After 4 d, samples were collected and analyzed by flow cytometry for lineage and FcγRII/III⁺ progenitor surface markers. Proliferation was assessed as a function of CellTrace Violet dye dilution after 7 d in culture. LK cells treated and expanded as above were purified by Gr-1 expression and analyzed by Western blot for MPO content. In a separate experiment, we measured STAT-3 phosphorylation and downstream gene expression in ST6Gal-1-treated and control LK exposed to 100 ng/ml G-CSF. (A) Quantification of total number of CD11b^hiGr-1^hi neutrophils per milliliter recovered after 4 d in culture (n = 6). P < 0.0001. (B) Representative plot of myeloid progenitor composition, with total GPs and MPs recovered after 4 d in culture. (C) Effects of rST6Gal-1 on cell proliferation. Light purple peaks represent initial intensity of CellTrace Violet staining. Percentages denote proportion of cells that have undergone only 1 cell division (n = 3). P < 0.0001. (D) Representative p-STAT-3 (Tyr705) blot after G-CSF treatment of LK cells. (E) Expression of several genes downstream of the G-CSF pathway was assessed by RT-qPCR analysis relative to β-2 microglobulin after 24 h cytokine treatment. (F) MPO protein content of Gr-1⁺ cells isolated from 7 d LK culture, normalized to β-actin. Values shown are relative to highest control sample (n = 3). **P < 0.01.

Figure 6.. ST6Gal-1 suppresses differentiation of human CD34⁺ cells in vitro.

Mobilized human CD34⁺ bone marrow was plated at a density of 0.5 × 10⁶ cells/ml and expanded in vitro in the presence of rhSCF, IL-3, and G-CSF. Before expansion, ST6Gal-1-treated cells were subjected to 3 h of ex vivo sialylation. Aliquots were removed approximately every 2 d and analyzed for cellularity and surface phenotype. (A) Total cellularity in wells receiving rST6Gal-1 vs. control (n = 5). (B) Total cells per well found to express either CD11b or CD34 surface markers as assessed by flow cytometry (n = 5). *P < 0.05; **P < 0.01; ***P < 0.001. (C and D) MPO-stained cytospins of cells collected at the termination of the experiment.