Immunomodulatory effect of bovine lactoferrin during SARS-CoV-2 infection

Abstract

Introduction: Lactoferrin (Lf) is an important immunomodulator in infections caused by different agents. During SARS-CoV-2 infection, Lf can hinder or prevent virus access to the intracellular environment. Severe cases of COVID-19 are related to increased production of cytokines, accompanied by a weak type 1 interferon response.

Methods: We investigated the influence of bovine Lf (bLf) in the immune response during SARS-CoV-2 infection in vitro and in vivo assays.

Results: Our results show a strong binding between bLf and TLR4/NF-κB in silico, as well as an increase in mRNA expression of these genes in peripheral blood mononuclear cells (PBMCs) treated with bLf. Furthermore, the treatment increased TLR4/TLR9 mRNA expression in infected K18-hACE2 mouse blood, indicating an activation of innate response. Our results show that, when bLf was added, a reduction in the NK cell population was found, presenting a similar effect on PD-1 in TCD4⁺ and TCD8⁺ cells. In the culture supernatant of PBMCs from healthy participants, bLf decreased IL-6 levels and increased CCL5 in COVID-19 participants. In addition, K18-hACE2 mice infected and treated with bLf presented an increase of serum pro-inflammatory markers (GM-CSF/IL-1β/IL-2) and upregulated mRNA expression of IL1B and IL6 in the lung tissue. Furthermore, bLf treatment was able to restore FTH1 levels in brain tissue.

Discussion: The data indicate that bLf can be part of a therapeutic strategy to promote the immunomodulation effect, leading to homeostasis during COVID-19.

Keywords: COVID-19; TLR4; bovine lactoferrin; cytokines; immunomodulation.

Figure 1

Cytokine levels in different tissues, showing the immunomodulatory effect of bLf in K18-hACE2 mice infected with SARS-CoV-2. (A) Experimental design; (B) serum levels of IFN-γ, IL-1β, GM-CSF, and IL-2; (C) IL6, TLR4, and TLR9 gene expression in whole blood; (D) CCL2, IL6, IFNB1, and FTH1 gene expression in brain tissue; (E) IL1B, CASP1, CCL2, IL6, and NOX1 gene expression in lung tissue. Kruskal–Wallis tests with Dunn’s post-hoc tests were applied to compare groups. *p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.00005. NC, negative control; PC, positive control (COVID-19); treated, treated with bLf.

Figure 2

Percentage of circulating immune cells from patients with COVID-19 and unexposed individuals, at different stages of the disease. (A) Gate strategy accessing the subpopulation of PBMCs by ex vivo immunophenotyping. (B) Percentage of total NK cells, (C) total monocytes, (D) total CD4⁺ T cells, and (E) total CD8⁺ T cells, obtained from unexposed individuals and patient with COVID-19. Percentage of obtained from unexposed individuals and patients with COVID-19 (**p < 0.01, and ****p < 0.0001).

Figure 3

In vitro effects of bLf on subpopulations of NK and T cells from unexposed and COVID-19 groups. (A) Percentage of NK^bright (CD3⁻CD56^highCD16⁻), (B) NKT(CD3⁺CD56⁺), (C) CD4⁺ activated T cells (CD69⁺), (D) exhausted CD4⁺ T cells (PD-1⁺), and (E) exhausted CD8⁺ T (PD-1⁺) cells upon stimulation with different bLf concentrations (1, 5, and 10 mg/mL), as well as unstimulated (mock). Levels of (F) IL-6, (G) CXCL8 (IL-8), and (H) CCL5 (RANTES) detected in supernatant obtained from cell stimulation conditions. *p < 0.05 and **p < 0.01.

Figure 4

Upregulated genes expression after in vitro bLf treatment of PBMCs in the acute phase of COVID-19. Expression levels of NFKB (A), IFIT1 (B), NCF4 (C), and TLR4 (D). Kruskal–Wallis tests with Dunn’s post-hoc tests were applied to compare groups. NC, negative control (unexposed); NT, no treatment (COVID-19); bLf, treatment with bLf.

Figure 5

Molecular docking of bLf with TLR4 and NF-κB. Structure of molecular docking pose of bLf with TLR4 (A) and NF-κB (B), showing residue interactions as well as bound types.