An engineered nano-liposome-human ACE2 decoy neutralizes SARS-CoV-2 Spike protein-induced inflammation in both murine and human macrophages

Abstract

Rationale: Macrophages are the frontline immune cells in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Angiotensin-converting enzyme 2 (ACE2) serves as the binding receptor to SARS-CoV-2 Spike glycoprotein for fusion and internalization into the human host cells. However, the mechanisms underlying SARS-CoV-2-elicited macrophage inflammatory responses remain elusive. Neutralizing SARS-CoV-2 by human ACE2 (hACE2) decoys has been proposed as a therapeutic approach to ameliorate SARS-CoV-2-stimulated inflammation. This study aims to investigate whether an engineered decoy receptor can abrogate SARS-CoV-2-induced macrophage inflammation. Methods: hACE2 was biotinylated to the surface of nano-liposomes (d = 100 nm) to generate Liposome-human ACE2 complex (Lipo-hACE2). Lentivirus expressing Spike protein (D614G) was also created as a pseudo-SARS-CoV-2 (Lenti-Spike). Liposome-hACE2 was used as a decoy receptor or competitive inhibitor to inhibit SARS-CoV-2 or Lenti-Spike-induced macrophage inflammation in vitro and in vivo. Results: Both SARS-CoV-2 and Lenti-Spike stimulated strong inflammatory responses by inducing the expression of key cytokine and chemokines, including IL-1β, IL-6, TNFα, CCL-2, and CXCL-10, in murine and human macrophages in vitro, whereas Lipo-hACE2 decoy abolished these effects in macrophages. Furthermore, intravenous injection of Lenti-Spike led to increased macrophage and tissue inflammation in wild type mice, which was also abolished by Lipo-hACE2 treatment. Mechanistically, Spike protein stimulated macrophage inflammation by activating canonical NF-κB signaling. RNA sequencing analysis revealed that Lenti-Spike induced over 2,000 differentially expressed genes (DEGs) in murine macrophages, but deficiency of IκB kinase β (IKKβ), a key regulator for NF-κB activation, abrogated Lenti-Spike-elicited macrophage inflammatory responses. Conclusions: We demonstrated that the engineered Lipo-hACE2 acts as a molecular decoy to neutralize SARS-CoV-2 or Spike protein-induced inflammation in both murine and human macrophages, and activation of the canonical IKKβ/NF-κB signaling is essential for SARS-CoV-2-elicited macrophage inflammatory responses.

Keywords: IKKβ/NF-κB signaling; Liposome-Human ACE2; SARS-CoV-2; Spike Protein; myeloid-specific IKKβ knockout.

Figure 1

Generation of liposome-hACE2 complex that prevents SARS-CoV-2-induced cell death. (A) Liposomes coated with human-ACE2 protein (Lipo-hACE2) were created via biotin-neutravidin bridge. Double signal from hACE2 absorbance (450nm OD) and rhodamine incorporated (λ^Ex 546 nm, λ^Em 568 nm) in 100nm lipoparticles confirmed the correct formation of the lipo-hACE2 decoy results are displayed as a percentage (n = 12; ***P < 0.001). (B) Vero E6 cells were infected with SARS-CoV-2 (2.5x10⁵ pfu/mL) alone or in combination with free-hACE2 protein (25 nM) or liposome-hACE2 (25 nM). Cell viability was measured after 48 h via AlamarBlue assay (n = 5; ***P < 0.001). (C) Schematic representation on how lipo-hACE2 inhibits SARS-CoV-2. Despite similar chemical affinity between free-hACE2 protein and Lipo-hACE2 for the SARS-CoV-2 spike proteins, Lipo-hACE2 also offers physical inhibition due to the liposomes size (100 nm) which may potentially enhance therapeutic effects. (D) Dose-response (5 to 50 nM) QPCR analysis was carried out to assess the lipo-hACE2 efficiency in terms of SARS-CoV-2 inhibition. Lipo-Control was used as negative control and free-hACE2 protein was used to compare inhibition efficiency.

Figure 2

SARS-CoV-2 Spike protein-induced inflammatory responses are inhibited by Liposome-hACE2 complex in murine macrophages. (A) Peritoneal macrophages were isolated from eight-week-old C57BL6/J wild-type (WT) mice. The cells were infected with Lentivirus expressing SARS-CoV-2 Spike protein (Lenti-Spike) (2.3x10⁷ pfu/mL) or control lentivirus for 1 h followed by treatment with 25 nM Lipo-hACE2 or control liposome for another 23 h. (B) Total RNA was extracted, and the expressions of inflammatory cytokines and chemokines were analyzed by QPCR (n = 5; ***P < 0.001).

Figure 3

Liposome-hACE2 complex ameliorates SARS-CoV-2-induced inflammatory responses in murine macrophages. (A) Peritoneal macrophages were isolated from eight-week-old C57BL6/J wild-type (WT) mice. The cells were infected with SARS-CoV-2 (2.5x10⁵ pfu/mL) or inactivated SARS-CoV-2 (control virus) for 1 h followed by treatment with 25 nM Lipo-hACE2 or control liposome for 23 h. (B) Total RNA was extracted and the expressions of inflammatory cytokines and chemokines were analyzed by QPCR (n = 5; *P < 0.05, **P < 0.01 and ***P < 0.001).

Figure 4

SARS-CoV-2 Spike protein-elicited macrophage inflammatory responses are attenuated by Liposome-hACE2 complex in vivo. (A) Eight-week-old male WT mice were injected IV with 100 µL of Lenti-spike (1.0x10⁶ pfu/mL) or control lentivirus followed by IV injection of 100 µL control liposome or Lipo-hACE2 (4 nM) 1 h later. After 24 h, mice were euthanized, and peritoneal macrophages were isolated for analysis. (B) Peritoneal macrophages were then lysated and total RNA was extracted. The expression of inflammatory cytokines and chemokines were analyzed by QPCR (n = 6; *P < 0.05, **P < 0.01 and ***P < 0.001).

Figure 5

Liposome-hACE2 complex ameliorates SARS-CoV-2 Spike protein-induced lung inflammation in vivo. (A) Eight-week-old male WT mice were injected IV with 100 µL of Lenti-spike (1.0x10⁶ pfu/mL) or control lentivirus followed by IV injection of 100 µL control liposome or Lipo-hACE2 (4 nM) 1 h later. After 24 h, mice were euthanized, and bronchoalveolar lavage fluid was collected for analysis. (B) Total RNA was extracted from bronchoalveolar lavage fluid, and the expression of inflammatory cytokines and chemokines were analyzed by QPCR (n = 6; *P < 0.05, **P < 0.01 and ***P < 0.001). (C) WT mice were injected IV with Lenti-spike or control lentivirus followed by IV injection of 100 µL control liposome or Lipo-hACE2 1 h later. After 24 h, mice were euthanized, and lung was collected for immunostaining. (D) Representative images of immunofluorescence staining of macrophage marker CD68 (top) and IL-6 (bottom) in the lung of WT mice. The nuclei were visualized with DAPI (blue) (scale bar, 100 µm).

Figure 6

SARS-CoV-2 Spike protein-induced cardiac inflammation is attenuated by Liposome-hACE2 complex in vivo. (A) Eight-week-old male WT mice were injected IV with 100 µL of Lenti-spike (1.0x10⁶ pfu/mL) or control lentivirus followed by IV injection of 100 µL control liposome or Lipo-hACE2 (4 nM) 1 h later. After 24 h, mice were euthanized, and hearts were collected for analysis. (B) Total RNA was extracted from the heart, and the expression of inflammatory cytokines and chemokines were analyzed by QPCR (n = 6; *P < 0.01, **P < 0.01 and ***P < 0.001). (C) Representative images of immunofluorescence staining of macrophage marker CD68 (top) and IL-6 (bottom) in the heart of WT mice. The nuclei were visualized with DAPI (blue) (scale bar, 100 µm).

Figure 7

Liposome-hACE2 suppresses SARS-CoV-2 Spike protein-induced inflammatory responses in human THP-1 macrophages. (A) Human THP‐1 monocytes were differentiated into macrophages by exposure to 100 ng/mL of phorbol‐12‐myristate‐13‐acetate (PMA) for 48 h. The differentiated macrophages were infected with Lentivirus expressing Spike protein (Lenti-Spike) (2.3x10⁷ pfu/mL) or control lentivirus for 1 h followed by treatment with 25 nM Lipo-hACE2 or control liposome for another 23 h. (B) Total RNA was extracted, and the expressions of inflammatory cytokines and chemokines were analyzed by QPCR (n = 3; *P < 0.05, **P < 0.01 and ***P < 0.001).

Figure 8

SARS-CoV-2 and Spike protein-induced inflammatory responses are blocked by Liposome-hACE2 complex in human peripheral blood mononuclear cells. (A) Human peripheral blood mononuclear cells (PBMC) were infected with Lentivirus expressing Spike protein (Lenti-Spike) (2.3x10⁷ pfu/mL) or control lentivirus for 1 h followed by treatment with 25 nM Lipo-hACE2 or control liposome for another 23 h. (B) Total RNA was extracted, and the expressions of inflammatory cytokines and chemokines were analyzed by QPCR (n = 3, *P < 0.05 and **P < 0.01). (C) Human PBMC were infected with SARS-CoV-2 (2.5x10⁵ pfu/mL) or inactivated SARS-CoV-2 (control virus) for 1 h followed by treatment with control liposome, 25 nM Lipo-hACE2, or 25 nM free-hACE2 for another 23 h. (D) Total RNA was extracted, and the expressions of inflammatory cytokines and chemokines were analyzed by QPCR (n = 3; *P < 0.05, **P < 0.01 and ***P < 0.001).

Figure 8

SARS-CoV-2 and Spike protein-induced inflammatory responses are blocked by Liposome-hACE2 complex in human peripheral blood mononuclear cells. (A) Human peripheral blood mononuclear cells (PBMC) were infected with Lentivirus expressing Spike protein (Lenti-Spike) (2.3x10⁷ pfu/mL) or control lentivirus for 1 h followed by treatment with 25 nM Lipo-hACE2 or control liposome for another 23 h. (B) Total RNA was extracted, and the expressions of inflammatory cytokines and chemokines were analyzed by QPCR (n = 3, *P < 0.05 and **P < 0.01). (C) Human PBMC were infected with SARS-CoV-2 (2.5x10⁵ pfu/mL) or inactivated SARS-CoV-2 (control virus) for 1 h followed by treatment with control liposome, 25 nM Lipo-hACE2, or 25 nM free-hACE2 for another 23 h. (D) Total RNA was extracted, and the expressions of inflammatory cytokines and chemokines were analyzed by QPCR (n = 3; *P < 0.05, **P < 0.01 and ***P < 0.001).

Figure 9

SARS-CoV-2 Spike protein induces macrophage inflammatory responses through IKKβ signaling in vitro. (A) Peritoneal macrophages were isolated from eight-week-old male IKKβ^F/F and IKKβ^ΔMye mice. Macrophages were infected with Lentivirus expressing SARS-CoV-2 Spike protein (Lenti-Spike) (2.3x10⁷ pfu/mL) or control lentivirus for 24 h. Total RNA was isolated for RNAseq analysis (n = 5). (B) Volcano plot of differential expression between Lenti-Spike and control treatment in macrophages of IKKβ^F/F and IKKβ^ΔMye mice. Colored dots represent the enriched (orange dots) or depleted (blue dots) differentially expressed genes (DEGs) with a false discovery rate (FDR) of < 1% and a fold change (FC) >3 as a cut-off threshold. (C) KEGG pathways significantly associated with the DEGs in control macrophages after Lenti-Spike infection. The P-values were computed by Fisher's exact test. The vertical dash line indicates the significance level of α = 0.01. The y-axis displays the KEGG pathways while the x-axis displays the P-values. (D) Geneset scores of the prioritized KEGG pathways. The geneset score was calculated using the FAIME algorithm. (E) Heatmap representation of DEGs involved in the pathways of “cytokine-cytokine receptor interaction”, “TNF signaling pathway”, “Antigen processing and presentation”, “NOB-like receptor signaling pathway”, “NF-kappa B signaling pathway”, and “Toll-like receptor signaling pathway”, “Cell adhesion molecules”, and “Chemokine signaling pathway”. Each row shows one individual gene and each column a biological replicate of mouse. Red represents relatively increased gene expression while blue denotes downregulation.