Effect of lactoferrin protein on red blood cells and macrophages: mechanism of parasite-host interaction

Abstract

Background: Lactoferrin is a natural multifunctional protein known to have antitumor, antimicrobial, and anti-inflammatory activity. Apart from its antimicrobial effects, lactoferrin is known to boost the immune response by enhancing antioxidants. Lactoferrin exists in various forms depending on its iron saturation. The present study was done to observe the effect of lactoferrin, isolated from bovine and buffalo colostrum, on red blood cells (RBCs) and macrophages (human monocytic cell line-derived macrophages THP1 cells).

Methods: Lactoferrin obtained from both species and in different iron saturation forms were used in the present study, and treatment of host cells were given with different forms of lactoferrin at different concentrations. These treated host cells were used for various studies, including morphometric analysis, viability by MTT assay, survivin gene expression, production of reactive oxygen species, phagocytic properties, invasion assay, and Toll-like receptor-4, Toll-like receptor-9, and MDR1 expression, to investigate the interaction between lactoferrin and host cells and the possible mechanism of action with regard to parasitic infections.

Results: The mechanism of interaction between host cells and lactoferrin have shown various aspects of gene expression and cellular activity depending on the degree of iron saturation of lactoferrin. A significant increase (P<0.05) in production of reactive oxygen species, phagocytic activity, and Toll-like receptor expression was observed in host cells incubated with iron-saturated lactoferrin when compared with an untreated control group. However, there was no significant (P>0.05) change in percentage viability in the different groups of host cells treated, and no downregulation of survivin gene expression was found at 48 hours post-incubation. Upregulation of the Toll-like receptor and downregulation of the P-gp gene confirmed the immunomodulatory potential of lactoferrin protein.

Conclusion: The present study details the interaction between lactoferrin and parasite host cells, ie, RBCs and macrophages, using various cellular processes and expression studies. The study reveals the possible mechanism of action against various intracellular pathogens such as Toxoplasma, Plasmodium, Leishmania, Trypanosoma, and Mycobacterium. The presence of iron in lactoferrin plays an important role in enhancing the various activities taking place inside these cells. This work provides a lot of information about targeting lactoferrin against many parasitic infections which can rule out the exact pathways for inhibition of diseases caused by intracellular microbes mainly targeting RBCs and macrophages for their survival. Therefore, this initial study can serve as a baseline for further evaluation of the mechanism of action of lactoferrin against parasitic diseases, which is not fully understood to date.

Keywords: cytotoxicity; lactoferrin; morphometric analysis; phagocytosis.

Figure 1

(A) Giemsa stain microscopy of red blood cells treated with different forms of lactoferrin at different concentrations (100×) and the untreated group. This experiment was repeated in triplicate. Morphological characteristics, ie, shape, size, and diameter, were the same in the different treated red blood cells compared with the untreated group. (B) Morphometric analysis of RBCs treated for 48 hours with the different proteins at a concentration of 40 µg/mL. No significant difference was found between the treated and untreated groups. Abbreviations: BLf, bovine lactoferrin; BLf Fe, bovine apolactoferrin; BLf Fe, iron-saturated bovine lactoferrin; BuLf, buffalo lactoferrin; BuLf Apo, buffalo apolactoferrin; BuLf Fe, iron-saturated buffalo lactoferrin; RBSs, red blood cells.

Figure 2

(A) ROS production in the differently treated groups of RBCs after 48 hours of incubation. (B) ROS production shown using bar diagrams. Each value is a replicate of three values. A significant fold increase *P<0.05 was found in the iron-treated groups when compared with the untreated group and significant P<0.05 difference was found within different groups, along with a significant *P<0.05 decrease in ROS production in the apolactoferrin-treated group compared with the untreated group. Abbreviations: Buff, buffalo; Apo, apolactoferritin; RBCs, red blood cells; ROS, reactive oxygen species; BLf, bovine lactoferrin; BLf Apo, bovine apolactoferrin; BLf Fe, iron-saturated bovine lactoferrin; BuLf, buffalo lactoferrin; BuLf Apo, buffalo apolactoferrin; BuLf Fe, iron-saturated buffalo lactoferrin.

Figure 3

(A) MTT viability assay showing percent viability of treated and untreated macrophages at 48 hours post-incubation. (B) Giemsa-stained macrophages after treatment with different proteins (100×). (C) Morphometric analysis of different groups of macrophages at 48 hours post-incubation. No significant difference was found between the various groups. Abbreviations: BLf, bovine lactoferrin; BLf Apo, bovine apolactoferrin; BLf Fe, iron-saturated bovine lactoferrin; BuLf, buffalo lactoferrin; BuLf Apo, buffalo apolactoferrin; BuLf Fe, iron-saturated buffalo lactoferrin.

Figure 4

(A) Significant fold increase in ROS production at increasing dose concentrations ranging from 10 to 50 µg/mL of the various lactoferrins. (B) Significant (P<0.005) fold increase in ROS production when used at 20 µg/mL compared with the untreated group and within the group (*P<0.05) shown by bar diagram. Each value is the replicate of three experimental values. *P<0.05; **P<0.005. Abbreviations: Lf, lactoferrin; ROS, reactive oxygen species; BLf, bovine lactoferrin; BLf Apo, bovine apolactoferrin; BLf Fe, iron-saturated bovine lactoferrin; BuLf, buffalo lactoferrin; BuLf Apo, buffalo apolactoferrin; BuLf Fe, iron-saturated buffalo lactoferrin.

Figure 5

Phagocytic capacity and invasion assay. (A) Flow cytometric analysis showing the pattern of phagocytic capacity in the different iron saturations of proteins confirmed by confocal microscopy (100×). (B) Phagocytic activity of treated and untreated macrophages shown in a bar diagram at 48 hours post-incubation. Significant **P<0.005 phagocytic intensity was found in iron-treated cells. (C) Invasion assay of Toxoplasma tachyzoites for the differently treated groups showed a significant difference **P<0.05 was found in the number of infected macrophages as highest number found in Fe treated cells (P<0.005) compared to untreated group and (*P<0.05) within different groups. Significance difference (*P<0.05) was found in phagocytic capacity of cells when comparison was done between Fe treated group. (D) Significant difference was found (*P<0.05) in the number of tachyzoites per macrophages when compared with untreated group and other groups. Abbreviations: BLf, bovine lactoferrin; BLf Apo, bovine apolactoferrin; BLf Fe, iron-saturated bovine lactoferrin; BuLf, buffalo lactoferrin; BuLf Apo, buffalo apolactoferrin; BuLf Fe, iron-saturated buffalo lactoferrin; FACS, fluorescence-activated cell sorting; Lf, lactoferrin.

Figure 6

Expression levels of the different genes. (A) All treated cells showed a significant difference (P<0.005) in MDR1 gene regulation (B) Survivin gene expression compared with the untreated group and other groups at 48 hours post-incubation. (C) & (D) All the treated cells showed elevated levels of TLR4 and 9 expression and significant high levels were observed in Fe-Lf treated cells when compared with untreated group and other groups. (E) The TLR antagonist achieved a 7–9-fold downregulation of expression in the different groups. A significant difference (P<0.05) was found between the iron-saturated lactoferrin-treated group compared with the other groups. *=P<0.05; **=P<0.005. Abbreviations: BLf, bovine lactoferrin; BLf Apo, bovine apolactoferrin; BLf Fe, iron-saturated bovine lactoferrin; BuLf, buffalo lactoferrin; BuLf Apo, buffalo apolactoferrin; BuLf Fe, iron-saturated buffalo lactoferrin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TLR, Toll-like receptor.

Figure 7

(A) Normal cell showing less TLR expression, less free radical production, and less phagocytic capacity. (B) Lactoferrin-treated cell showing better phagocytosis, more TLR signaling, more free radical production, and less MDR1 gene expression after treatment with iron-saturated lactoferrin. Abbreviations: ds RNA, double-stranded RNA; LPS, lipopolysaccharide; Lf, lactoferrin; P-gp, P-glycoprotein; TLR, Toll-like receptor.