Neuraminidase inhibitors rewire neutrophil function in vivo in murine sepsis and ex vivo in COVID-19

Abstract

Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo, treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections.

Keywords: COVID-19; Oseltamivir; SARS-CoV-2; Zanamivir; neuraminidase; neutrophil; sepsis; sialic acid.

Figure 1.. LPS stimulates NEU activity in human leukocytes.

Neuraminidase isolated from Clostridium perfringens (CpNEU) was used to validate the NEU activity assay. CpNEU (0.012 UI) was added in 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-MU-NANA (0.025 mM) was added and the fluorescent substrate was read 3 min after at 37 °C (A). The area under the curve (AUC) values are shown in B. Total leukocytes resuspended in HBSS were added in a plate on ice and 4-MU-NANA substrate (0.025 mM) was added followed by the addition of medium, LPS (1 μg/mL), LPS plus Oseltamivir (100 μM) or LPS plus Zanamivir (30 μM). The fluorescent substrate was read 3 min after at 37 °C (C). Raw data were subtracted from the control group containing only HBSS (medium) and expressed as AUC values (D). Whole blood containing 1 × 10⁶ leukocytes from healthy donors were stimulated or not with LPS (1 μg/mL, 90 min, 37 °C, 5% CO₂), LPS plus Oseltamivir (100 μM), or LPS plus Zanamivir (30 μM). Total leukocytes (1 × 10⁶) were incubated with CpNEU (10 mU, 60 min, 37 °C, 5% CO₂), CpNEU plus Oseltamivir (100 μM), or CpNEU plus Zanamivir (30 μM). Leukocytes were stained with MAL-II to detect α2–3 sialic acids (E-F; K-L), with SNA to detect α2–6 sialic acids (G-H; M-N) or PNA to detect galactosyl (β–1,3) N-acetylgalactosamine (I-J; O-P). The MFI was analyzed on CD66b⁺/CD15⁺ cells using the gate strategies shown in Supplementary Fig. 1. *P< 0.05; **P < 0.01; ***P < 0.001. This figure is representative of three independent experiments (n= 3–6) and data are shown as mean ± SEM. LPS = lipopolysaccharide; CpNEU = neuraminidase; MAL-II = Maackia amurensis lectin II; SNA = Sambucus nigra lectin; PNA = peanut agglutinin.

Figure 2.. LPS increases phagocytosis and killing of E. coli in a NEU-dependent manner.

Whole blood from healthy donors containing 1 × 10⁶ leukocytes were exposed (37 °C, 5% CO₂) or not to LPS (1 μg/mL, 90 min), LPS plus Oseltamivir (100 μM), or LPS plus Zanamivir (30 μM) (A-C; G-H; K). Total leukocytes (1 × 10⁶) were exposed or not to CpNEU (10 mU, 60 min, 37 °C, 5% CO₂), CpNEU plus Oseltamivir (100 μM), or CpNEU plus Zanamivir (30 μM) (D-F; I-J; L) and the phagocytosis and killing assays were performed. Leukocytes were incubated with E. coli pHrodo BioParticles^® (100 μg/mL) for 60 min at 37 °C to assess phagocytosis in viable CD66b⁺/CD15⁺ cells (A-F) (as gated in Supplementary Fig. 1). Live E. coli was used to evaluate phagocytosis by light microscopy or to assess the killing by leukocytes. Cells were stimulated as described above and 1 ×10⁶ leukocytes were incubated at 37 °C with E. coli (1 ×10⁶ CFU) for 90 min for phagocytosis or for 180 min for killing assays. The percentage of cells with ingested bacteria (G; I) and the number of bacterial particles per cell (H; J, ≥3 particles per cell) were evaluated. The killing of E. coli was evaluated by spreading 10 μL of supernatant (extracellular killing) or 10 μL of the intracellular content in agar medium and the CFU were counted. Killing E. coli was expressed as the rate of fold change compared to the unprimed (untreated) cells (L). Symbols represent individual donors and data are shown as mean ± SEM from pooled data of two to three independent experiments (n = 3–12). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.. LPS-induced human neutrophil response involves NEU activity.

Whole blood from healthy donors containing 1 × 10⁶ leukocytes were stimulated or not with LPS (1 μg/mL, 90 min, 37 °C, 5% CO₂), LPS plus Oseltamivir (Osel, 100 μM), LPS plus Zanamivir (Zana, 30 μM), or LPS plus MAL-II (1 μg/mL, MAL-II promotes steric hindrance at the NEU cleavage site and prevent sialic acid cleavage). Leukocytes were marked with MAL-II (A-C) or with the cell activation markers CD62L (D-F) and CD66b (G-I). After red blood cells lysis leukocytes were incubated with 5 μM CM-H2DCFDA fluorescent probe for 15 min. PMA (10 μM) was used to stimulate ROS production for 10 min (J-L). Supplementary Fig. 5 showed ROS production in additional control groups. The MFI was analyzed on CD66b⁺ cells using the gate strategies shown in Supplementary Fig. 1. Isolated neutrophils were treated with Osetamivir (100 μM) or Zanamivir (30 μM) 1 h before the stimulus with LPS (10 μg/mL) for 4 h. The concentration of NETs was evaluated by MPO-DNA PicoGreen assay on supernatants of cells (M). Symbols represent individual donors and data are shown as mean ± SEM from pooled data of two to three independent experiments (n = 7) except for F and M that was made once with n=3. ***P < 0.001; **P < 0.01. CM-H2DCFDA = 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester; PMA = phorbol 12-myristate 13-acetate; ROS = reactive oxygen species; NETs = neutrophil extracellular traps; PMN = polymorphonuclear leukocytes.

Figure 4.. Oseltamivir enhanced mice survival in K. pneumoniae-induced sepsis.

Sepsis was induced by intratracheal administration of K. pneumoniae and mice were randomly treated (starting 6 hr after infection, 12/12 hr, PO, n=20) with saline or Oseltamivir phosphate (10 mg/kg) and survival rates were monitored for 144 hr (A). In similar set of experiments, septic mice (n=6–7) were treated 6 hr after infection with a single dose of Oseltamivir phosphate (10 mg/kg, PO) and mice were euthanized 24 hr after infection to determine the number of neutrophils (B) and CFUs (C), and levels of TNF (D) and IL-17 (E) in BAL. Plasma levels of TNF (F), IL-17 (G), AST (H), ALT (I), ALP (J) and total bilirubin (K) were also evaluated 24 hr after infection. The amount of surface α2–3 sialic acids were assessed by MAL-II staining in SSC^high/Gr-1^high cells in BAL and analyzed 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; *P < 0.01; ***P < 0.001. Sham = sham-operated mice; Osel = Oseltamivir; AST = alanine aminotransferase; ALT = aspartate aminotransferase; ALP = alkaline phosphatase.

Figure 5.. High expression of NEU1 in cell types from COVID-19 patients.

(A) Gene expression of NEU1, NEU3 and NEU4 across cell types in healthy donors and moderate or critical COVID-19 patients. Size of the circle is proportional to the percentage of cells expressing the reported genes at a normalized expression level higher than one. (B) UMAP analysis colored-coded by cell types in nasopharyngeal/pharyngeal swabs samples from healthy donors and COVID-19 patients. (C) Normalized expression of NEU1 overlaid on the UMAP spaces.

Figure 6.. Oseltamivir and Zanamivir decrease neutrophil activation and increase sialic acid levels in active, but not convalescent neutrophils from COVID-19 patients.

Whole blood from healthy donors (n= 10), severe COVID-19 patients (n= 6) or convalescent COVID-19 patients (n= 8) were treated or not with Oseltamivir (100 μM) or Zanamivir (30 μM) and total leukocytes were stained with the cell activation markers CD62L (A and E), CD66b (B and F) or the lectins MAL-II (C, D and G), SNA (H) and PNA (I). Immunofluorescence (D) was carried out using biotinylated MAL-II followed by streptavidin Alexa Fluor 555 conjugate. Three different healthy donors (controls) and severe Covid-19 patients were used (magnification 100×). Blood samples from healthy donors (n = 7) were incubated for 2 h (37 °C, 5% CO₂) with 7% of fresh plasma from healthy donors, severe or convalescent COVID-19 patients or with 7% of heat-inactivated plasma from severe COVID-19 patients in the presence or absence of Oseltamivir (100 μM) or Zanamivir (30 μM). Levels of CD66b (J), surface α2–3-Sia (MAL-II) (K), and ROS production (L, M) were assessed by FACS. The MFI was analyzed on CD66b⁺/CD15⁺ cells using the gate strategies shown in Supplementary Fig. 1. Symbols represent individual donors and data are shown as scatter dot plot with line at median from pooled data of two to seven independent experiments. The statistical significance between the groups was assessed by ANOVA followed by a multiple comparisons test of Tukey. The accepted level of significance for the test was P<0.05. * was significantly different when compared with Untreated Severe COVID-19; # was significantly different when compared with Untreated Heat-Inactivated Plasma from Severe COVID-19. Osel = Oseltamivir; Zana = Zanamivir.