Neuraminidase is a host-directed approach to regulate neutrophil responses in sepsis and COVID-19

Abstract

Background and purpose: Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Because pathogen-derived neuraminidase (NEU) stimulates neutrophils, we investigated whether host NEU can be targeted to regulate the neutrophil dysregulation observed in severe infections.

Experimental approach: The effects of NEU inhibitors on lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients also was carried out. The effects of oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models.

Key results: Oseltamivir and zanamivir constrained host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with matrix metalloproteinase 9 (MMP-9). Inhibition of MMP-9 prevented LPS-induced NEU activity and neutrophil response. In vivo, treatment with oseltamivir fine-tuned neutrophil migration and improved infection control as well as host survival in peritonitis and pneumonia sepsis. NEU1 also is highly expressed in neutrophils from COVID-19 patients, and treatment of whole-blood samples from these patients with either oseltamivir or zanamivir reduced neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage.

Conclusion and implications: These findings suggest that interplay of NEU1-MMP-9 induces neutrophil overactivation. In vivo, NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.

Keywords: COVID-19; SARS-CoV-2; metalloproteinase-9; neuraminidase; neutrophil; oseltamivir; sepsis; sialic acid; zanamivir.

FIGURE 1

Lipopolysaccharide (LPS) stimulates neuraminidase (NEU) activity in human neutrophils. NEU from Clostridium perfringens (CpNEU) was used to validate the NEU activity assay. CpNEU (0.012 UI) was added to a 96‐well flat‐bottom dark plate on ice in the presence or absence of oseltamivir phosphate (100 μM) or zanamivir (30 μM). Next, the substrate 4‐methylumbelliferyl‐N‐acetyl‐α‐d‐neuraminic acid (4‐MU‐NANA) (0.025 mM) was added, and the fluorescent resulting product read at 37°C for 55 min (a). The area under the curve (AUC) values are shown in (b). Isolated neutrophils (1 × 10⁶) from healthy donors (n = 6) were stimulated or not with CpNEU (0.012 UI, 60 min, 37°C, 5% CO₂) in the presence or absence of oseltamivir (100 μM) or zanamivir (30 μM). Neutrophils were then stained with Maackia amurensis lectin II (MAL‐II) to detect α‐2,3 sialic acids (c), Sambucus nigra lectin (SNA) to detect α‐2,6 sialic acids (d) or peanut agglutinin (PNA) to detect galactosyl (β‐1,3) N‐acetylgalactosamine (e) using flow cytometry analysis. Cells (0.5 × 10⁶; n = 5) resuspended in HBSS were added to a plate on ice, and 4‐MU‐NANA substrate (0.025 mM) was added followed by the addition of medium and LPS (1 μg·ml⁻¹, 60 min, 37°C, 5% CO₂) in the presence or absence of oseltamivir (100 μM) or zanamivir (30 μM) (f). The AUC values are shown in (g). The property of LPS in stimulating endogenous NEU from human isolated neutrophils (n = 6) was evaluated using different concentrations of oseltamivir (10, 30, 100 and 300 μM) or zanamivir (1, 10, 30 and 100 μM). Sialic acid content was assessed using staining with MAL‐II (h), SNA (i) and PNA (j), and cell activation was evaluated by staining CD62L (k), CXCR2 (l) and CD11b (m) by flow cytometry. The MFI or the percentage (%) in relation to the unstimulated control group was analysed on CD66b⁺/CD15⁺ cells using the gate strategies shown in Figure S1.^* P < 0.05.^#Significantly different compared to LPS‐stimulated groups. Symbols represent individual donors, and data are shown as mean ± SEM from experiments conducted with at least five different individuals.

FIGURE 2

Lipopolysaccharide (LPS) increases phagocytosis and killing of E. coli in an NEU‐dependent manner. Whole blood from healthy donors (n = 6) containing 1 × 10⁶ leukocytes was incubated or not with LPS (1 μg·ml⁻¹, 90 min, 37°C, 5% CO₂) in the presence or absence of oseltamivir (100 μM) or zanamivir (30 μM). The E. coli pHrodo BioParticles® (100 μg·ml⁻¹) were used for 60 min at 37°C to assess phagocytosis by flow cytometry. Representative histograms were used to show the effect of oseltamivir (a) and zanamivir (b) or by the MFI (c) in viable CD66b⁺/CD15⁺ cells (as gated in Figure S1). Live E. coli was used to assess phagocytosis by light microscopy or to investigate bacterial killing by leukocytes. Cells were stimulated as described above, and 1 × 10⁶ leukocytes were incubated at 37°C with E. coli (1 × 10⁶ colony‐forming units [CFU]) for 90 min for phagocytosis (n = 6) or for 180 min for the killing assay (n = 5). The percentage of cells with ingested bacteria (d) and the number of bacterial particles per cell (e, ≥3 particles per cell) were evaluated. The killing of E. coli was studied by spreading 10 μl of supernatant (extracellular killing) or 10 μl of the intracellular or membrane adhered content in agar medium for CFU counting. The killing was expressed as the rate of fold change compared to the unprimed (untreated) cells (f). Symbols represent individual donors, and data are shown as mean ± SEM from experiments conducted with at least five different individuals.^* P < 0.05

FIGURE 3

Lipopolysaccharide (LPS)‐induced NEU1 activity is dependent on matrix metalloproteinase 9 (MMP‐9) in human neutrophils. Isolated neutrophils from healthy donors (n = 5) were stimulated or not with LPS (1 μg·ml⁻¹, 90 min, 37°C, 5% CO₂), and cytospin slides containing 0.3 × 10⁶ cells were used to detect NEU1 membrane staining (red) and 4′,6‐diamidino‐2‐phenylindole (DAPI) for the fluorescent deoxyribonucleic acid (DNA) stain (blue) by indirect immunofluorescence (a). A similar approach was carried out showing NEU1 in the membrane of isolated neutrophils (1 × 10⁶) by FACS (c). NEU1–MMP‐9 surface interaction (yellow) was assessed using the Duolink® proximity ligation assay (PLA) (d). Merged representative photos (magnification 100×) were captured from at least three different fields and analysed using ImageJ® 1.53J software. MFI was assessed in each captured field (each point corresponds to the mean of a different donor) and normalised by the number of DAPI‐positive cells (b, e). LPS‐stimulated neutrophils (1 × 10⁶, n = 5) in the presence or absence of Maackia amurensis lectin II (MAL‐II) (1 μg·ml⁻¹, MAL‐II promotes steric hindrance at the NEU cleavage site and prevents sialic acid cleavage) or the selective MMP‐9 inhibitor (5 nM) was added 30 min prior to LPS incubation. Neutrophil‐derived neuraminidase activity was assessed using 4‐methylumbelliferyl‐N‐acetyl‐α‐d‐neuraminic acid (4‐MU‐NANA) substrate (f). The area under the curve (AUC) values are shown in (g). Cells (n = 5) were also then stained in the conditions described above with MAL‐II to detect α‐2,3 sialic acids (h), Sambucus nigra lectin (SNA) to detect α‐2,6 sialic acids (i) or peanut agglutinin (PNA) to detect galactosyl (β‐1,3) N‐acetylgalactosamine (j). The cell activation markers CD66b (k), CD62L (l), CXCR2 (m) and CD11b (n) were evaluated by flow cytometry. The percentage (%) in relation to the unstimulated group was analysed on CD66b⁺/CD15⁺ cells using the gate strategies shown in Figure S1.^* P < 0.05. Symbols represent individual donors, and data are shown as mean ± SEM from experiments conducted with at least five different individuals.

FIGURE 4

Lipopolysaccharide (LPS)‐ or opsonized zymosan‐induced ROS production by human neutrophils is dependent on NEU/matrix metalloproteinase 9 (MMP‐9) activity. Isolated neutrophils (1 × 10⁶) from healthy donors (n = 5) were stimulated or not with LPS (1 μg·ml⁻¹, 90 min, 37°C, 5% CO₂) in the presence or absence of oseltamivir (100 μM) or zanamivir (30 μM). Experiments also were conducted using pretreatment with Maackia amurensis lectin II (MAL‐II) (1 μg·ml⁻¹) or the selective MMP‐9 inhibitor (5 nM) added 30 min prior to LPS incubation. Cells were washed and resuspended in HBSS containing luminol and stimulated or not with phorbol 12‐myristate 13‐acetate (PMA) (0.1 μg·ml⁻¹), opsonized zymosan (OZ; 0.5 μg·ml⁻¹) or N‐formylmethionine‐leucyl‐phenylalanine (fMLF) (1 μM) for chemiluminescence (CL) detection. Kinetic analysis of luminol CL was expressed as integrated total counts for oseltamivir and zanamivir (a, d, g) or followed the pretreatment with MAL‐II or MMP‐9i (b, e, h). The area under the curve (AUC) values are shown in (c), (f) and (i).^* P < 0.05. Data are shown as mean ± SEM from experiments performed in duplicates with five different healthy donors (n = 5).

FIGURE 5

Oseltamivir (Osel) enhanced mice survival in Klebsiella pneumoniae‐induced sepsis. Sepsis was induced by intratracheal administration of K. pneumoniae, and mice were randomly treated (starting 6 h after infection, 12/12 h, PO, n = 20) with saline or Osel phosphate (10 mg·kg⁻¹), and survival rates were monitored for 144 h (a). In a similar set of experiments, septic mice (n = 5) were treated 6 h after infection with a single dose of Osel phosphate (10 mg·kg⁻¹, PO), and mice were euthanized 24 h after infection to determine the number of neutrophils (b), colony‐forming units (CFUs) (c) and the levels of TNF (d) and IL‐17 (e) in bronchoalveolar lavage (BAL). Plasma levels of TNF (f), IL‐17 (g), aspartate aminotransferase (AST) (h), alanine aminotransferase (ALT) (i), alkaline phosphatase (ALP) (j) and total bilirubin (k) were evaluated 24 h after infection. The amount of surface α‐2,3 sialic acids was assessed by Maackia amurensis lectin II (MAL‐II) staining in SSC^high/GR‐1^high cells in BAL and analysed by FACS, as shown by the representative histograms (l) and MFI (m); dotted line = unstained cells. The results are expressed as percent of survival, mean or median (only for FACS data) ± SEM.^* P < 0.05. Sham, sham‐operated mice

FIGURE 6

High expression of NEU1 in cell types from COVID‐19 patients. Gene expression of NEU1, NEU3 and NEU4 across cell types in healthy donors, moderate or critical COVID‐19 patients. The size of the circle is proportional to the percentage of cells expressing the reported genes at a normalized expression level higher than one (a). UMAP analysis colour coded by cell types in nasopharyngeal/pharyngeal swab samples from healthy donors and COVID‐19 patients (b). Normalized expression of NEU1 overlaid on the UMAP spaces (c).

FIGURE 7

Oseltamivir (Osel) and zanamivir (Zana) decrease neutrophil activation and increase sialic acid levels in active, but not convalescent neutrophils from COVID‐19 patients. Whole blood from healthy donors and severe, critical ICU or convalescent COVID‐19 patients was treated or not with Osel (100 μM) or Zana (30 μM), and total leukocytes were stained with the cell activation markers CD62L (a, j), CD66b (b, k) or the lectins Maackia amurensis lectin II (MAL‐II) (c, f, l), Sambucus nigra lectin (SNA) (m) and peanut agglutinin (PNA) (n) (n = 6–7 per group). Isolated neutrophils (0.5 × 10⁶) from healthy controls (n = 5) and severe (n = 6) or critical ICU COVID‐19 patients (n = 5) were added in a 96‐well flat‐bottom dark plate on ice. Next, the substrate 4‐MU‐NANA (0.025 mM) was added, and the resulting fluorescent product read at 37°C for 30 min (d). The area under the curve (AUC) values are shown in (e). Cytospin slides containing 0.3 × 10⁶ unstimulated neutrophils from healthy donors (n = 5) or severe COVID‐19 patients (n = 5) were used to detect α‐2,3 sialic acids (f) or NEU1 (h) membrane staining (red) as well as 4′,6‐diamidino‐2‐phenylindole (DAPI) for the fluorescent deoxyribonucleic acid (DNA) stain (blue) by indirect immunofluorescence. Representative images (1000×) were captured from at least three different fields and analysed using ImageJ 1.53J software. MFI was assessed in each captured field (each point corresponds to the mean of a different donor) and normalized by the number of DAPI‐positive cells (g, i). Total reactive oxygen species (ROS) production was assessed by FACS in isolated neutrophils at the basal state from healthy donors (n = 6) or severe COVID‐19 patients (n = 6) using the CM‐H2DCFDA probe (o). Total blood from healthy donors (n = 7) was incubated (2 h, 37°C, 5% CO₂) with 7% fresh plasma obtained from healthy controls and severe or convalescent COVID‐19 patients. Incubations were carried out in the presence or absence of 7% of heat‐inactivated plasma from severe COVID‐19 patients under pretreatment or not with Osel (100 μM) or Zana (30 μM). Levels of CD66b (p), surface α‐2,3‐Sia (MAL‐II) (q) and ROS production (r) were assessed by FACS. The MFI was analysed on CD66b⁺/CD15⁺ cells using the gate strategies shown in Figure S1. Symbols represent individual donors, and data are shown as scatter dot plots with lines at median from pooled data of two to seven independent experiments. The accepted level of significance for the test was P < 0.05.^*Significantly different when compared with untreated severe COVID‐19.^#Significantly different when compared with untreated heat‐inactivated plasma from severe COVID‐19

FIGURE 8

Oseltamivir (Osel) treatment inhibits mouse hepatitis virus 3 (MHV‐3) replication and inflammation‐associated injury in the lungs of wild‐type MHV‐3‐infected mice. C57BL/6 mice (n = 6) infection was carried out under anaesthesia with ketamine (50 mg·kg⁻¹) and xylazine (5 mg·kg⁻¹) by intranasal inoculation with culture medium (mock) or the murine coronavirus MHV‐3 (3 × 10³ plaque‐forming unit [PFU]). After 24 h of inoculation, mice were treated or not with Osel phosphate (10 mg·kg⁻¹) or 0.5% sodium carboxymethyl cellulose (CMC) (v/v) starting 24 h after infection, 12/12 h, PO. Three days after infection, all animals were euthanized, and lungs were collected for further analysis. Viral load was assessed in lung lysates by plaque assay, and results were expressed as log₁₀ PFU·g⁻¹ of lung tissue (a). Neutrophil infiltration into the lungs was determined indirectly by myeloperoxidase (MPO) activity using a colorimetric assay, and results were expressed as mean optical density (mOD)·mg⁻¹ of lungs (b). Histological lung analysis was done using tissues stained with haematoxylin and eosin (H&E), and results were expressed by representative images (40× magnification; scale: 50 μm) from the different groups (c) and by a histopathological score analysis (d). The results are expressed as mean ± SEM.^* P < 0.05