To validate that there is an inverse relationship between the naturally occurring ST6Gal-1 in circulation and the need to produce new inflammatory cells during demand granulopoiesis, we subjected naïve, native C57BL/6 mice to a challenge with NTHI directly into the airways. NTHI elicits a Type 1 immune response in the airways that is dominated by neutrophilic infiltration in the initial phase. Circulatory ST6Gal-1 was monitored in these animals by assessing the enzymatic ability to form α2,6-sialyl linkages onto Gal(β1,4)GlcNAc acceptor substrate. NTHI exposure generated a pronounced but transient depression of circulatory ST6Gal-1 activity to ~30% of baseline levels at 7 h (Figure 1, left). In contrast, other sialyltransferase activities in the blood, specifically those forming the α2,3-sialyl structures on Gal(β1,4)GlcNAc and mediated by the sialyltransferases ST3Gal-3,−4, or−6, were not altered upon NTHI exposure (Figure 1, right).

We reported previously that insufficient circulatory ST6Gal-1 levels result in accelerated de novo granulocyte accumulation (1–3). Here, we validated this observation in the NTHI model of acute airway inflammation. The globally ST6Gal-1 null mouse, St6gal1-KO, and the St6gal1-dP1 mouse with deficiency only in the circulatory pool of extracellular ST6Gal-1 were examined. As summarized in Figure 2A, both St6gal1-dP1 and St6gal1-KO mice had exaggerated neutrophil accumulation in the BALF compared to wild-type animals 18 h after NTHI challenge. Moreover, nearly identical ~1.5-fold augmented accumulation of neutrophils was observed in both ST6Gal-1 deficit models that were generated independently by different strategies. Neutrophils dominated the inflammatory cell infiltrate into the airway in the acute inflammatory response to NTHI. On average, 0.8 × 10⁶ neutrophils were recoverable in the bronchial alveolar lavage fluid (BALF) at 18 h in native C57BL/6 (wild-type) animal, and neutrophils comprised >75% of the total recovered BALF cells (Figure 2B). This result, which is more fully presented in Supplemental Table 1A, strongly supports the conclusion that insufficiency in circulatory ST6Gal-1, rather than intracellular Golgi-ER bound ST6Gal-1, was the factor driving excess neutrophil accumulation in the airways in acute inflammation.

Enhanced efficiency in recruitment of granulocytes into the airway of St6gal1-dP1 mice compared to wild-type animals did not drive the exaggerated neutrophil accumulation. Neutrophils were isolated from St6gal1-dP1 and C57BL/6 mice, differentially labeled with either PKH26 or PKH67 fluorescent dyes, pooled, and intravenously transferred into wild-type recipients. The recruitment of the adoptively infused neutrophils into NTHI-induced acute airway was monitored as ratios of the differentially dyed cells by flow cytometry. Figure 2C shows that the ratios of the St6gal1-dP1 to wild-type neutrophils recovered in the BALF were not changed from the original input ratios. The data confirm that circulatory ST6Gal-1 deficiency resulted in a more severe acute airway inflammatory response to NTHI challenge, and that the mechanism is more robust granulopoiesis rather than altered efficiencies of cell recruitment to the inflamed lung.

We observed in murine genetic models of circulatory ST6Gal-1 deficiency more pronounced peritonitis and airway acute inflammation elicited by sterile agents such as LPS (1, 22). Chronic elevation of circulatory ST6Gal-1 by subcutaneous implantation of a B16 melanoma engineered to release ST6Gal-1 partially alleviated the sterile agent induced acute airway (1). Here, we posit that an infection-driven acute inflammation can also be attenuated by raising circulatory ST6Gal-1. We further posit that infusion of pure, recombinant ST6Gal-1 (rST6G), resulting only in temporary elevation of blood ST6Gal-1 activity can be effective against infection-driven inflammation. To explore the potential therapeutic value of rST6G, this and all other following experiments were performed in the wild-type C57BL/6 mouse. A single bolus of rST6G in its present formulation, when infused into wild-type animals at baseline was rapidly cleared from the bloodstream in <1 h (see Supplemental Figure 1A). Despite the rapid clearance, a single rST6G infusion resulted in a striking decline in granulopoietic parameters within the bone marrow, with ~40% decrease in colony forming units in granulocyte (G), Monocyte (M) and GM-CFUs 7 h later (Figure 3A). Total marrow cellularity diminished overall by ~25%, resulting mostly from a >2-fold reduction in marrow neutrophils and a slight reduction in B220-positive cells, the two most-abundant marrow cell populations (Figure 3B). Blood differentials revealed an almost 50% reduction in total white cell counts, accountable by the diminution of circulatory lymphocyte numbers that are the major white cell constituents in the blood (Figure 3C). Curiously, circulatory neutrophils in the naïve wild-type animals were not altered, although at baseline only a minor (10–12%) percentage of the overall circulating white cells are granulocytes. The complete blood differential counts are presented in Supplementary Table 1B.

To assess the anti-inflammatory efficacy of systemically administered rST6G, NTHI-challenged C57BL/6 mice received 2 intravenous infusions of rST6G, the first at 2 h after receiving NTHI, and a booster at 10 h. Two intravenous rST6G infusions were used as a precaution, because we observed rapid clearance of the current formulation of rST6G from the blood (Supplemental Figure 1A). The two infusions 5, 8 h apart resulted in circulatory ST6Gal-1 activity that was 2-fold over baseline at 16h after the initial rST6G infusion (Supplemental Figure 1B). In cohorts receiving rST6G, BALF neutrophil counts were reduced by ~50% (Figure 4B). Only a slight (10%) reduction in overall BALF leukocyte counts was observed, due to a ~2.5-fold increase in recruited macrophage. Though the increase in recruited macrophage numbers was unexpected, it is noteworthy that animals with genetic ST6Gal-1 deficit had ~33% decrease in recruited macrophage numbers in the airway following NTHI exposure (see Supplemental Table 1A). Circulating blood counts, monitored at the time BALF was recovered, showed no differences between rST6G and sham animals. This is not unexpected, since circulating neutrophilia was noted to be extremely transient and limited to the first few hours after a peripheral acute challenge, including peritonitis by LPS or thioglycollate (2), airway eosinophilia by OVA to sensitized mice (3), and acute airway inflammation by LPS (30). Blinded histopathologic evaluation disclosed a consistent reduction in pulmonary inflammation among animals treated with rST6G. Compared to animals receiving saline, the rST6G treated group showed smaller inflammatory cuffs around bronchovascular bundles and fewer inflammatory cells within alveolar walls and alveolar spaces (Figures 4C,D). The histopathologic scoring is summarized in Supplemental Table 2. Most unexpectedly, the rST6G-treated group had strikingly lowered levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the BALF. In fact, inflammatory cytokines were close to or below reliable assay detection limits in BALF from animals that received rST6G, when compared to easily quantifiable levels in sham treated animals (Figure 5).

To gain mechanistic insight into the blunted inflammatory cytokines released in the airways by rST6G treatment, despite the apparently paradoxical elevation of airway macrophage, one of the principal cell types along with epithelial cells responsible for the release of inflammatory cytokines (31, 32), we examined the response of primary airway macrophages. Airway macrophages isolated from the BALF of resting wild-type C56BL/6 mice were stimulated ex vivo with heat-killed NTHI in the presence or absence of rST6G. A reduction of NTHI-dependent release of TNF-α and IL-6 production was observed in the rST6G-treated macrophages (Figure 6A). This effect was not unique to airway macrophages, as bone marrow derived macrophages treated with rST6G also had a 3.5-fold reduction in TNF-α (Figure 6B), Interestingly, IL-10 production by NTHI stimulated BM macrophages was elevated in the rST6G treated cells, pointing tantalizingly to a possible additional pathway by which rST6G can mitigate acute inflammation. In these ex vivo experiments, 0.1 mM CMP-Sia was also included. We have observed that rST6G to have effect on suppressing macrophage activity ex vivo, even without added sialic acid donor substrate. Possibly, the leakage of sialic acid donor substrate from neighboring dying cells might be sufficient. However, we also observed that addition of CMP-Sia has the benefit of diminishing variability. In vivo, sialic acid donor substrate is believed to be supplied by activating platelets (23, 33).

In an earlier report, we showed that systemic ST6Gal-1 dampens granulopoiesis in the marrow by extrinsic modification of the hematopoietic progenitor cells through the attachment of α2,6-linked sialic acid residues, which can be monitored by the lectin, SNA (Sambucus nigra agglutinin) (22). Here, cell surface α2,6-sialylation status of airway macrophage recovered in the BALF of animals challenged in vivo with NTHI was assessed for changes in SNA reactivity. The data show a pronounced increase in cell surface SNA reactivity in cells from animals treated with rST6G, compared to saline treated animals (Figure 6C). This observation strongly suggests that rST6G introduced into systemic circulation were able to affect the pulmonary macrophages and blunt the release of inflammatory cytokines during an NTHI-elicited acute airway response.

The St6gal1-dP1 and St6gal1-KO mice strains were in the C57BL/6J background as described previously (2). Unless otherwise stated, C57BL6/J mice between 7 and 10 weeks of age were used, and both sexes were equally represented. Roswell Park Institute of Animal Care and Use Committee (IACUC) approved maintenance of animals and all procedures used under protocol 1071M. There is no involvement of human subjects or clinical specimens; ethics committee review is not required according to the local and national guidelines.

rST6G is the recombinant secretory form of rat ST6Gal-1 where the catalytic domain was generated as a fusion protein encoding the following: NH₂-signal sequence – 8x His tag – Avi tag – GFP – TEV protease cleavage site – ST6GAL1 catalytic domain – COOH (47). The construct was expressed in HEK293 cells; the recombinant protein was harvested and purified from the medium. The ST6Gal-1 catalytic domain was proteolytically released by TEV protease digestion and further purified (47, 48). Sialyltransferase assays were carried out as described previously (23).

A frozen glycerol stock of NTHI strain 1479 (clinical isolate from a COPD exacerbation) was streaked on chocolate-agar plates, and single colonies were grown in a liquid culture of brain–heart infusion media supplemented with 10 μg/ml hemin and 10 μg/ml β-NAD (Sigma). After 3–4 h of culture in a 37°C shaking incubator, OD₆₀₀ was determined to dilute the required number of CFU to 2 × 10⁸ CFU/ml in PBS. Bacteria were pelleted in microcentrifuge tubes at 13,000 × g for 10 min and washed twice in PBS. To initiate acute NTHI-mediated inflammation, mice were anesthetized by isoflurane inhalation, and 50 μl (1 × 10⁶ CFU) live NTHI diluted in PBS was use for oropharyngeal instillation using a 200-μl sterile pipette tip.

rST6G was injected i.v. (750 μg/CC in PBS) 2 and 14 h after NTHI exposure. Same volume of PBS was injected to control mice. After 18 h, mice were sacrificed by injection (i.p.) of two 0.5 ml Avertin (2.5 gr 2,2,2, Tribromethanol, 5 ml 2-methyl-2-butanol in 200 ml distilled water). Bronchoalveolar lavage (BAL) was performed post euthanization by opening the thoracic cavity to expose the trachea, which was cannulated with a 22-gauge i.v. catheter. PBS (750 μL) was injected and withdrawn from the lung two times using a tuberculin syringe. For cytokine assays, BAL fluid (200 μl) or serum (50 μl) was subjected to Luminex 100 multiplex assays using a capture bead system developed by Luminex Corporation (Austin, TX, USA). For pathohistologic evaluations, lungs were excised and fixed in 10% formaldehyde in PBS, paraffin embedded, sectioned, and stained with H&E. Lung pathology was evaluated by a board certified pulmonary pathologist blinded to the identity of the slides.

Flow cytometry was performed using anti-CD45 (hematopoietic cells), anti-Ly6G (neutrophils), anti-B220(B cells), anti-CD3 (T cells), anti-F4/80 (macrophage) anti-CD11c (dendritic cells) antibodies and SNA (Sambucus nigra lectin, Vector Laboratories, Peterborough, UK). All reagents were purchased from BioLegend (San Diego, CA). Cells were analyzed using BD LSRII flow cytometer (Becton Dickinson Immunocytometry Systems). For colony forming cell assays, marrow nucleated cells in a volume of 0.1 ml were plated in 0.9 ml of methylcellulose medium (MethoCult 3534, STEMCELL Technologies) in duplicate and placed in humidified incubator with 5% CO₂ at 37°C. Colonies containing at least 50 cells were counted 7 days after incubation.

Bone marrow cells were collected from hind-limbs of mice, either St6gal1-dP1 or C57BL/6 wild-type, re-suspended in RBC lysis buffer (0.8% NH₄Cl, 0.1 mM EDTA buffered with KHCO₃ to pH 7.4), washed and re-suspended in phosphate-buffered saline (PBS) with 0.5% BSA or fetal bovine serum and 2 mM EDTA, and then passed through a 100-μm cell strainer (BD Biosciences). Cells were centrifuged and re-suspended in the same buffer (up to 2 × 10⁸ cells/ml), and 50 μl/ml of biotinylated antibodies (anti-cKit. anti-B220, anti-CD3, anti-TER119, anti-CD5) was added to the cell suspension. Partial neutrophil enrichment was accomplished by negative selection using magnetic microparticles according to the manufacturer's protocol (STEMCELL Technologies, Vancouver, British Columbia, Canada). More than 60% of selected cells were neutrophils as verified by flow cytometry. To apply labeling with PKH26 and PKH67 (Sigma Chemical, St Louis, MO), cells were washed in RPMI medium (without serum), and 10⁷ cells were re-suspended in 1 mL Diluent C (Sigma) and rapidly added to 1 mL of 4 μM PKH26 or PKH-67. The cells were incubated at 25°C for 5 min, terminated by the addition of 2.5% fetal calf serum. After labeling, the cells were washed twice with cold PBS and counted by hemocytometer. Differentially labeled donor cells were mixed 1:1 immediately before infusion into recipient animals. A small fraction of combined cells was labeled with neutrophil marker (anti-Ly6G antibody) and saved for measuring donor WT/dP1 neutrophil ratios. Each recipient received pooled cells consisting of 10⁷ cells intravenously from each labeled group 2 h after NTHI BAL was performed 18 h after NTHI.

Testing for differences between mean values was determined using either Students' T-Test or two-way ANOVA with post-test comparisons in Graph Pad Prism 6 software (La Jolla, CA). p < 0.05 is considered significant.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.