Oral recombinant human or mouse lactoferrin reduces Mycobacterium tuberculosis TDM induced granulomatous lung pathology

Abstract

Trehalose 6'6-dimycolate (TDM) is the most abundant glycolipid on the cell wall of Mycobacterium tuberculosis (MTB). TDM is capable of inducing granulomatous pathology in mouse models that resembles those induced by MTB infection. Using the acute TDM model, this work investigates the effect of recombinant human and mouse lactoferrin to reduce granulomatous pathology. C57BL/6 mice were injected intravenously with TDM at a dose of 25 μg·mouse^-1. At day 4 and 6, recombinant human or mouse lactoferrin (1 mg·(100 μL)^-1·mouse^-1) were delivered by gavage. At day 7 after TDM injection, mice were evaluated for lung pathology, cytokine production, and leukocyte populations. Mice given human or mouse lactoferrin had reduced production of IL-12p40 in their lungs. Mouse lactoferrin increased IL-6 and KC (CXCL1) in lung tissue. Increased numbers of macrophages were observed in TDM-injected mice given human or mouse lactoferrin. Granulomatous pathology, composed of mainly migrated leukocytes, was visually reduced in mice that received human or mouse lactoferrin. Quantitation of granulomatous pathology demonstrated a significant decrease in mice given human or mouse lactoferrin compared with TDM control mice. This report is the first to directly compare the immune modulatory effects of both heterologous recombinant human and homologous mouse lactoferrin on the development of TDM-induced granulomas.

Keywords: human lactoferrin; lactoferrine de souris; lactoferrine humaine; mouse lactoferrin; trehalose dimycolate; tréhalose dimycolate.

Fig. 1

Effect of oral lactoferrin on lung weight index (LWI). C57BL/6 mice injected with trehalose 6'6-dimycolate (TDM) were treated with human or mouse lactoferrin (LF) on days 4 and 6 after injection. Mice were sacrificed on day 7. Whole lung mass was measured and compared with the mass of the mouse. Data are plotted as box and whiskers, marking the range, the 1st and 3rd quartiles, and the mean.

Fig. 2

Lung tissue cytokine environment. C57BL/6 mice injected with trehalose 6'6-dimycolate (TDM) were treated with human or mouse lactoferrin (LF) at days 4 and 6 after injection. Mice were sacrificed on day 7. A section of lung was collected, homogenized, and incubated with media for 4 h. Supernatants were analyzed by ELISA. Data are plotted as box and whiskers, marking range, the 1st and 3rd quartiles, and the mean; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 3

Leukocyte populations in lung tissue on day 7 after injection with trehalose 6'6-dimycolate (TDM). C57BL/6 mice injected with TDM were treated with human or mouse lactoferrin (LF) on days 4 and 6 after injection. Mice were sacrificed on day 7. A section of lung was collected and digested into single cell suspensions. Cells were stained for CD3, CD4, CD8, NK1.1, CD11b, and GR-1. Data are plotted as box and whiskers, marking range, the 1st and 3rd quartiles, and the mean; *, p < 0.05.

Fig. 4

Lung histology for trehalose 6'6-dimycolate (TDM) induced mice after treatment with lactoferrin. C57BL/6 mice injected with TDM were treated with human or mouse lactoferrin (LF) on days 4 and 6 after injection. Mice were sacrificed on day 7. A section of lung was collected, fixed in 10% formalin, and stained with H&E. Lung sections were visualized at 20x. Images are representative of 10–14 lung sections across 3 experimental repeats. [Colour online.]

Fig. 5

Quantitation of granulomatous structures in lung sections treated with or without lactoferrin. C57BL/6 mice injected with trehalose 6'6-dimycolate (TDM) were treated with human or mouse lactoferrin (LF) on days 4 and 6 after injection. Mice were sacrificed on day 7. A section of lung was collected, fixed in 10% formalin, and stained with H&E. Lung sections were visualized at 20x. Image analysis to determine the percentage of lung area occupied by granulomatous structures was conducted with ImageJ.