Short-Term Effect of SARS-CoV-2 Spike Protein Receptor-Binding Domain-Specific Antibody Induction on Neutrophil-Mediated Immune Response in Mice

Abstract

Vaccination protects against COVID-19 via the spike protein receptor-binding domain (RBD)-specific antibody formation, but it also affects the innate immunity. The effects of specific antibody induction on neutrophils that can cause severe respiratory inflammation are important, though not completely investigated. In the present study, using a mouse model mimicking SARS-CoV-2 virus particle inhalation, we investigated neutrophil phenotype and activity alterations in the presence of RBD-specific antibodies. Mice were immunized with RBD and a week after a strong antibody response establishment received 100 nm particles in the RBD solution. Control mice received injections of a phosphate buffer instead of RBD. We show that the application of 100 nm particles in the RBD solution elevates neutrophil recruitment to the blood and the airways of RBD-immunized mice rather than in control mice. Analysis of bone marrow cells of mice with induced RBD-specific antibodies revealed the increased population of CXCR2⁺CD101⁺ neutrophils. These neutrophils did not demonstrate an enhanced ability of neutrophil extracellular traps (NETs) formation compared to the neutrophils from control mice. Thus, the induction of RBD-specific antibodies stimulates the activation of mature neutrophils that react to RBD-coated particles without triggering excessive inflammation.

Keywords: SARS-CoV-2 infection; antibodies; mouse model; neutrophils; receptor-binding domain.

Figure 1

RBD–specific antibody induction. (A) The scheme of i.p. injections of RBD to mice (black arrows) and the time points of blood collection (red arrows). (B,C) The levels of IgM (B), serum dilution 1:100, and IgG (C), serum dilution 1:1000, two weeks after the last 15 µg RBD injection (3 × 15 µg) and a week after the last 50 µg RBD injection (3 × 15 µg + 3 × 50 µg). (D) The strategy of T− and B−cells detection in peripheral blood of the mice. (E) The ratio of B−cells to T−cells a week after the last RBD injection. (F–H) The levels of periphery blood IgG1 (F), IgG2a (G) serum dilution 1:1000, and IgA (H), serum dilution 1:100, in mice that were injected with RBD (gray bars, black circles) or in the control mice that were injected with PBS (open bars, black circles), and in the intact mice (NM, open bars, open circles) a week after the last RBD injection. The representative data are shown (n ≥ 4 mice per group). A significant difference between the indicated group and the intact mice was detected using Mann–Whitney test *: p ≤ 0.05.

Figure 2

Neutrophil–mediated response to the application of 100 nm particles in the RBD solution. (A) The scheme of the RBD i.p. injections and o.ph. application of 100 nm particles in RBD solution to mice (black arrows). Two and 24 h after the o.ph application, blood was collected; 24 h after the application, BAL, lung and bone marrow were collected (red arrows). (B) The strategy of the neutrophil identification among blood cells or among myeloid cells. (C–E) The percentages of myeloid cells (C), neutrophils from myeloid cells (D) and neutrophils from live blood cells (E) 2 and 24 h after the o.ph. application of 100 nm particles in the RBD solution to the RBD—(gray bars, black circles) or PBS–injected mice (open bars, black circles); the intact mice NM (open bars, open circles). (F) The level of angiotensin II in the peripheral blood sera of mice injected with RBD (gray bars, black circles) or PBS (open bars, black circles) 2 and 24 h after the o.ph. application of 100 nm particles in the RBD solution, and the intact mice—NM (open bars, open circles). The data are shown as medians and interquartile range (i.q.r.) for n ≥ 4 mice per group. A significant difference between the indicated group and the intact mice was detected using the Mann–Whitney test *: p ≤ 0.05.

Figure 3

Identification and quantitative analysis of neutrophils in the airways and the lung vessels. (A) The representative three-dimensional image of the lung of mouse that received i.p. RBD injections and the o.ph. application of 100 nm particles in the RBD solution. The large vessels (CD106, magenta), the airways (Streptavidin, grayscale), and 100 nm particles agglomerates (FluoSpheres, red) are represented via volume rendering. Scale bar 1000 µm. (B) The higher magnification of the region boxed in (A) demonstrating the vessel (magenta) and CD11b⁺ cells (light blue) as the frontal (left image) and the lateral (right image) projections. Scale bar 100 µm. (C) The image of the region boxed in (B) showing the vessel and CD11b⁺ cells via surface rendering. Scale bar 40 µm. (D,E) Representative three-dimensional images of the regions of conducting airway mucosa of the mouse with induced RBD-specific antibodies (D) and of the PBS-injected mouse (E) 24 h after the application of 100 nm particles in the RBD solution. 100 nm particles (FluoSpheres, red), epithelial and smooth muscle cells (phalloidin, grayscale), neutrophils (Ly6G, light blue) and CD11c⁺ cells (green) are represented as the frontal (upper images) and the lateral (lower images) projections. Scale bar 30 µm. (F,G) The quantitative analysis of neutrophils in the airway mucosa (F) or BAL (G) of mice that were injected with RBD (gray bars, black circles) or with PBS (open bars, black circles) 24 h after the application of 100 nm particles in the RBD solution, and in intact mice (NM, open bars, open circles). (H,I) The levels of IgA (H) and IgG (I) in the BAL fluids of mice that were injected with RBD (gray bars, black circles) or with PBS (open bars, black circles) 24 h after the application of 100 nm particles in RBD solution, and in the intact mice (open bars, open circles). The data are shown as medians and i.q.r. for n ≥ 4 mice per group. A significant difference between the indicated group and the intact mice was detected using the Mann–Whitney test *: p ≤ 0.05.

Figure 4

RBD–specific antibody induction effect on the bone marrow neutrophil activation and maturation. (A) The strategy of CXCR2⁺ and CD101⁺ neutrophil detection in the bone marrow of the mice. (B–D) The percentages of neutrophils (B), CXCR2⁺ neutrophils (C), and CXCR2⁺CD101⁺ neutrophils (D) in the bone marrow of mice that were injected with RBD (gray bars, black circles), PBS (open bars, black circles) 24 h after the application of 100 nm particles in the RBD solution, and intact mice (open bars, open circles). The data are shown as medians and i.q.r. for n ≥ 4 mice per group. A significant difference between the indicated group and the intact mice or between the indicated groups was detected using the Mann–Whitney test *: p ≤ 0.05.

Figure 5

NET formation by bone marrow neutrophils from mice with RBD–specific antibodies. (A,B) The representative images of neutrophils yielded from the bone marrow of the PBS–injected mouse 3 h after the incubation in the presence (B) or without (A) of 5 µm of A23187. The neutrophils were stained with Cell mask (magenta), SytoxGreen (green), and Hoechst (blue). Scale bar 10 µm. (C) The quantitative analysis of the extracellular nucleotide levels 3 h after the incubation with A23187 of neutrophils from the bone marrow of mice injected with RBD (gray bars, black circles), PBS (open bars, black circles) or intact mice (open bars, open circles). Data are shown as medians and i.q.r. for n ≥ 4 mice per group. A significant difference between the indicated group and the intact mice or between the indicated groups was detected using the Mann–Whitney test *: p ≤ 0.05. (D–F). The representative images of nuclei (Hoechst, left images), citrullinated histone H3 (CytH3, middle images), and co-localization (merged, right images) are presented for the A23187–treated (D,E) and untreated neutrophils (F), yielded from RBD–injected (D) or PBS-injected (E,F) mouse 24 h after the application of 100 nm particles in the RBD solution. Scale bar 20 µm.