Recombinant Sialyltransferase Infusion Mitigates Infection-Driven Acute Lung Inflammation

Abstract

Inappropriate inflammation exacerbates a vast array of chronic and acute conditions with severe health risks. In certain situations, such as acute sepsis, traditional therapies may be inadequate in preventing severe organ damage or death. We have previously shown cell surface glycan modification by the circulating sialyltransferase ST6Gal-1 regulates de novo inflammatory cell production via a novel extrinsic glycosylation pathway. Here, we show that therapeutic administration of recombinant, bioactive ST6Gal-1 (rST6G) mitigates acute inflammation in a murine model mimicking acute exacerbations experienced by patients with chronic obstructive pulmonary disease (COPD). In addition to suppressing proximal neutrophil recruitment at onset of infection-mediated inflammation, rST6G also muted local cytokine production. Histologically, exposure with NTHI, a bacterium associated with COPD exacerbations, in rST6G-treated animals revealed consistent and pronounced reduction of pulmonary inflammation, characterized by smaller inflammatory cuffs around bronchovascular bundles, and fewer inflammatory cells within alveolar walls, alveolar spaces, and on pleural surfaces. Taken together, the data advance the idea that manipulating circulatory ST6Gal-1 levels has potential in managing inflammatory conditions by leveraging the combined approaches of controlling new inflammatory cell production and dampening the inflammation mediator cascade.

Keywords: ST6Gal-1; airway; extrinsic glycosylation; infection; inflammation; sialylation.

Figure 1

Transient depression of circulatory ST6Gal-1 accompanies acute airway inflammation. Live NTHI bacteria (10⁶ CFU / animal) were delivered by oropharengeal instillation. Blood was collected at the times shown after instillation. Sialyltransferase activities in the sera were measured by following the transfer of CMP-[3H]Sia to Galβ1–4GlcNAc-O-Bn (LacNAc). The Siaα2,6 product formed by ST6Gal-1 (Left), was separated from Siaα2,3 product formed by various ST3Gal transferases (Right) using SNA-agarose chromatography.

Figure 2

More severe neutrophilic acute airway inflammation in animals with ST6Gal-1 deficiency. Wild type C57BL/6 (WT), ST6Gal1-dP1 (dP1), and ST6Gal1-KO (KO) mice were exposed to 10⁶ CFU of live NTHI bacteria by oropharengeal instillation. Eighteen hours later, the bronchoalveolar lavage fluid (BALF) was collected, Leukocyte number was counted, and leukocyte composition was determined by flow cytometry. (A) shows the total numbers of neutrophils recovered from the BALF of NTHI-instilled animals, showing greater neutrophilic inflammation in dP1 and KO, compared to WT (1.6 and 2.0-fold, respectively). ^* p < 0.05 for indicated comparisons. (B), top shows the cellular composition of WT BALF, consisting predominantly of neutrophils (83.5%). Macrophage (10.5%), dendritic cells (DC, 3.8%), T- (1%), and B- (0.5%) cells, with a minor constituent of epithelial cells as defined by EpCAM (0.7%). (B), bottom, shows dP1 BALF composition, which was essentially identical to WT BALF in percentage cellular contribution from the assessed cell types. (C) Neutrophils from the marrows of WT and dP1 mice were isolated by negative selection. The cells were stained with one of the two distinct membrane dyes (red PKH-26 and green PKH-67), mixed in an ~1:1 ratio and injected into 3–4 WT recipients 2 h after NTHI challenge. In the top panel, the initial PKH67-labeled WT/ PKH26-labeled dP1 neutrophils ratio was 1.30. In the bottom panel, PKH26-WT/PKH67-dP1 neutrophil donor ratio was 1.04. At 18 h, this neutrophil-fluorescence ratio was again measured in cells obtained from the BALF. No difference in airway recruitment was noted for dP1 neutrophils compared to WT neutrophils.

Figure 3

Intravenous rST6G infusion depresses myelopoiesis and alters inflammatory cell availability. WT mice receiving either a single 300 μg bolus of recombinant ST6Gal-1 (rST6G) or saline (PBS) were sacrificed 7 h later. Bone marrow cells from hind limbs and peripheral blood were isolated and analyzed as follows. (A) presents marrow progenitor clonogenic activity for granulocyte/monocyte (GM), granulocyte (G), or monocyte (M) progenitor colonies. The combined total colony formed is also shown (Total). Saline- (open bars) and rST6G-treated mice (n = 9 each group) were used with 4 × 10⁴ marrow cells were plated in Methocult M3534 to promote growth of myeloid progenitors for 10 days. ^*p < 0.01 rST6G compared to PBS. (B) summarizes the overall bone marrow cellularity of PBS (round symbols) and rST6G-treated (square symbols) animals, where each symbol denotes one animal. Total bone marrow cellularity (Total), and neutrophil and B cell numbers are shown. ^*p < 0.01. (C) Summarizes white cell counts in the blood as total white blood cell (WBC) count and differential count for lymphocyte (Lymph), neutrophil (Neu), monocyte, eosinophil and basophil (MEB). PBS (n = 8) or rST6G-treated animals (n = 9) were used. ^*p < 0.01.

Figure 4

Acute airway inflammation induced by NTHI exposure is mitigated by rST6G infusion. (A) shows the intervention protocol where each animal received two 300 μg rST6G or saline/sham injections spaced 8 h apart with the first injection being at 2 h after NTHI challenge. Animals were sacrificed 18 h later and assessed for pulmonary inflammation. (B) Inflammatory cell accumulation in the BALF of the rST6G- and sham- (PBS) treated animals showing total BALF cells (Total), neutrophil (Neutr), and macrophage accumulation (Macs). ^*p < 0.01. (C,D) Show the lung pathology of saline and rST6G-treated animals, respectively at 18 h.

Figure 5

Inflammatory cytokine release during acute airway inflammation is suppressed by rST6G infusion. C57BL/6 wild-type animals were challenged with NTHI and subjected to the rST6G or sham (PBS) treatment protocol as outlined in Figure 4A. The bronchial alveolar lavage fluids (BALF) were analyzed for cytokines by Luminex 100 multiplex assays. Dashed lines and the shaded boxed regions represent the reliable lower assay limit of detection for each cytokine. Many of the values obtained, especially for the IL-10 assays and the rST6G-treated cohorts for IL-1β values, were zero. For these, a default low value of “1” was assigned in order to calculate the p value. ^*p < 0.001.

Figure 6

Inflammatory cytokine release by macrophage was attenuated by ST6Gal-1. (A) Macrophages were recovered from the BALF of 3 wild-type C57BL/6 mice (at rest). The pooled cells were plated into 5 replicate but identical wells for each determination. Groups of 5 macrophage wells were exposed ex vivo to 10⁵ CFU/ml heat-killed NTHI either in the absence or presence (20 μg/ml) of rST6G and CMP-Sia (100 μM) for 18 h. TNF-α and IL-6 released into the media was measured next day. ^*p < 0.001. (B) Bone marrow–derived macrophages were generated from marrow cells of C57BL/6 WT animals. The identically seeded cells, in groups of 5 wells per treatment, were exposed to heat-killed NTHI in the absence (control) or presence of rST6G (20 μg/ml) and CMP-Sia (100 μM) and incubated overnight (37°C and 5% CO₂). TNF-α and IL-10 released into the media were assessed by ELISA. ^*p < 0.001 based on concentration values from five separate wells for each condition. All data are representative of six separate experiments. (C) BALF macrophage was recovered from C57BL/6 WT animals 18 h after oropharyngeal challenge with NTHI using the protocol in Figure 4A, either with rST6G (rST6G) or sham (PBS). Cell surface sialylation was measured using the α2,6-sialic acid-specific lectin, FITC conjugated Sambucus nigra agglutinin (SNA) by flow cytometry, as shown.