Lactoferrin modulation of mycobacterial cord factor trehalose 6-6'-dimycolate induced granulomatous response

Abstract

The immune system responds to tuberculosis (TB) infection by forming granulomas. However, subsequent immune-mediated destruction of lung tissue is a cause of significant morbidity and contributes to disease transmission. Lactoferrin, an iron-binding glycoprotein, has demonstrated immunomodulatory properties that decrease tissue destruction and promote T(H)1 immune responses, both of which are essential for controlling TB infection. The cord factor trehalose 6,6'-dimycolate (TDM) model of granuloma formation mimics many aspects of TB infection with a similar histopathology accompanied by proinflammatory cytokine production. C57BL/6 mice were injected intravenously with TDM. A subset of mice was given 1 mg of bovine lactoferrin 24 h post-TDM challenge. Lung tissue was analyzed for histological response and for the production of proinflammatory mediators. C57BL/6 mice demonstrated a granuloma formation that correlated with an increased production of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α,) IL-12p40, interferon-gamma (IFN-γ), and IL-10 protein. Mice treated with lactoferrin postchallenge had significantly fewer and smaller granulomas compared with those given TDM alone. Proinflammatory and T(H)1 cytokines essential to the control of mycobacterial infections, such as TNF-α and IFN-γ, were not significantly different in mice treated with lactoferrin. Furthermore, the anti-inflammatory cytokines IL-10 and transforming growth factor-β were increased. A potential mechanism for decreased tissue damage observed in the lactoferrin-treated mice is proposed. Because of its influence to modulate immune responses, lactoferrin may be a useful adjunct in the treatment of granulomatous inflammation occurring during mycobacterial infection.

Figure 1

Lactoferrin modulation of TNF-α, IL-6, IL-12p40, and TGF-β production by BMM in response to TDM-coated beads. Cells were treated with 3 µm BSA- or TDM-coated beads alone at a ratio of 10:1, or with 100 µg/ml lactoferrin (LF) at the same time as bead stimulation, or with 100 µg/ml lactoferrin 3 hours after bead addition. Values were measured by ELISA and expressed as mean pg/ml with standard deviation (SD). * p<0.05 compared to treatment with TDM-coated beads alone.

Figure 2

Granulomatous response to TDM in lactoferrin treated mice. A) Mice were challenged with 25 µg TDM prepared in a water-in-oil emulsion. One mg of lactoferrin or transferrin (not shown) was administered 24 hours post-TDM challenge. Hematoxylin and eosin staining, magnification 100×. B) Number of lung granulomas per mm² and the size of the granulomas (µm²). Data are expressed as the mean ± SD. * p<0.05 with comparisons made to the mice administered the TDM emulsion.

Figure 3

Production of pro-inflammatory mediators in mice challenged with TDM and treated with lactoferrin. The levels of TNF-α, IL-1β, IL-6, IL-12p40, and IFN-γ in lung tissue were determined by ELISA. Data are shown as the mean with standard deviation. N = 8 mice per group, per time point. *p<0.05 with comparison to the TDM emulsion treated mice.

Figure 4

Lactoferrin induced production of anti-inflammatory cytokines in TDM challenged mice. The levels of IL-10 and TGF-β were quantified in lung tissue homogenates by ELISA. Data are expressed as the mean with standard deviation. N = 8 mice per group, per time points indicated. *p<0.05, comparisons made to the TDM-treated mice.

Figure 5

Lactoferrin modulation of BMM cytokine production in response to MTB infection. BMM were infected with MTB Erdman using a MOI of 1:1. 100 µg/ml of bovine lactoferrin was added at the same time as infection or 3 hours after infection. The levels of TNF-α, IL-6, IL-12p40, and TGF-β were measured in the filtered supernatants 24 and 72 hours after infection by ELISA. Data are shown as the mean and SD. *p<0.05, comparisons are to cells infected with MTB alone.