Lactoferrin as a natural immune modulator

Abstract

Lactoferrin, an iron-binding glycoprotein, is a cell-secreted mediator that bridges innate and adaptive immune function in mammals. It is a pleiotropic molecule that directly assists in the influence of presenting cells for the development of T-helper cell polarization. The aim of this review is to provide an overview of research regarding the role of lactoferrin in maintaining immune homeostasis, in particular as a mediator of immune responses to infectious assault, trauma and injury. These findings are critically relevant in the development of both prophylactic and therapeutic interventions in humans. Understanding these particular effects of lactoferrin will provide a logical framework for determining its role in health and disease.

Figures

Fig. 1. LF inhibits oxidative stress-induced apoptosis

Lactoferrin was assessed for activity to limit apoptosis as defined by activation of caspase-3 (A) or Annexin V presentation (B). U937 cell pellets were examined post-treatment with glucose oxidase (GO, 500 ng/mL), with or without lactoferrin (LF). Enzymatic reactions were performed with appropriate caspase substrate [45]. Caspase activity was determined by measuring the change in absorbance at 405 nm (A). Alternatively, cells were treated with GO alone or in the presence of lactoferrin (125 or 250 μg/mL) or with z-DEVD-fmk (N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoro-methylketone) a caspase–3 inhibitor of GO-induced Annexin V binding (B). Cells were then stained with Annexin V-FITC as described [204]. The mean fluorescence for 12,000 cells from three or more independent experiments are shown, expressed as ±SEM.

Fig. 2. Effect of lactoferrin on clearance of E. coli from circulation

Mice were challenged with a lethal dose of enterogenic bacteria (2×108 organisms, intravenous injection). The level of organisms within whole blood was measured 5 h post infection. Treatment of mice with LF (10 mg/mouse) 24 h prior to E. coli challenge (Lethal Dose, LD100) resulted in 100 fold faster clearing of bacteria from circulation, when compared with controls. E. coli + PBS (phosphate buffered saline, open); HSA (human serum albumin, closed); LF (lactoferrin, hatched). **p<0.05

Fig. 3. Effect of lactoferrin on the clearance of E. coli from various organs

Mice were challenged with a lethal dose of enterogenic bacteria (2×108 organisms, intravenous injection). Number of organisms was measured in lungs, spleens, livers and kidneys 5 h post infection. Treatment with LF (10 mg/mouse) 24 h prior to E. coli challenge (LD100) decreased the number of organisms recovered in spleens, livers and kidneys over 10 fold, and in lungs over 1000 fold, when compared with PBS and/or HSA controls. **p<0.05.

Fig. 4. Bovine lactoferrin pre-treatment leads to decreased proinflammatory responses from mice infected with E.coli

BALB/c mice were pre-treated with bovine lactoferrin at 1 mg/mouse (intravenous) or 5 mg/mouse (intraperitoneal) or remained untreated controls. After 4 h, mice were infected with a sub-lethal dose (~5×108 CFU/mouse) of E.coli SM105 (intravenous). At day 4 post-infection, blood was collected for analysis of cytokine production of IL-6 (A) and TNF-α (B) by ELISA. * p<0.05 compared to the control group not treated with lactoferrin.

Fig. 5. Lactoferrin pre-treatment protects host gut integrity against sub-lethal challenge with E. coli

BALB/c mice challenged with a sub-lethal dose of E.coli SM105 demonstrate severe jejunal tissue destruction at 4 days post infection (A). Parenchyma from mice pretreated with lactoferrin (1 mg/mouse, intravenous, 4 hours prior to challenge) remained intact (B), with no demonstration of tissue damage Jejunal tissue was collected and fixed with 10% formalin, paraffin embedded, sectioned, and stained with H&E. Visualized at 100×.

Fig. 6. Effect of lactoferrin on host protection against MRSA infection

BALB/c mice were pre-treated with bovine lactoferrin (1 mg/mouse, intravenous) or remain untreated. After 2 h, mice were infected with MRSA (strain Mu50, American Type Tissue Culture, intaperitoneal, 1×109 CFU/mouse). Mice were monitored for survival (A). At various time points, blood was collected for analysis of bacterial load (B) and cytokine production (C,D) by ELISA. * p<0.05 compared to the no lactoferrin control group.

Fig. 7. Lactoferrin decreases proinflammatory cytokines from BCG infected macrophages

Bone marrow derived macrophages were infected with BCG (MOI 10: 1) without (white) or with (black) bovine lactoferrin (100 μg/mL). At 72 h, supernatants were collected and analyzed by ELISA for TNF-α, IL-6, and IL-1β. * p<0.05, *** p<0.001.

Fig. 8. BCG infected BMDCs increase CD62L and decrease CD44 when cultured with bovine lactoferrin

Bone marrow derived dendritic cells (>90% CD11c+) (5×105 cells/mL) were cultured with BCG (MOI 10: 1) with or without bovine lactoferrin (100 mg/mL) or remained uninfected. After 72 h, cells were collected and stained for expression of CD62L-FITC (A) or CD44-FITC (B).

Fig. 9. Lactoferrin decreases T-cell cytokines from Con A stimulated splenocytes

Non-adherent splenocytes (2×106 cells/mL) were isolated from C57BL/6 mice. Production of IFN-γ (A) and IL-2 (B) were measured by ELISA after 72 h stimulation with Con A (2 μg/mL) in the presence of increasing concentration of bovine or human lactoferrin (1, 10, 100 μg/mL). * p<0.05

Fig. 10. Mice immunized with BCG and lactoferrin demonstrate diminished inflammation and destructive pulmonary histopathology upon challenge with virulent MTB

C57BL/6 mice (6 per group) demonstrated reduced histological manifestation of disease in the BCG/lactoferrin immunized groups at day 65 post challenge. The top panels depict histopathology from a non-immunized control and is compared to mice immunized and boosted with BCG vaccine (middle panels). The lactoferrin adjuvant immunized mice (bottom panels) revealed striking reduction in granulomas with evidence of lymphocytic clusters and contained focal pockets of inflamed monocytes. Staining with H&E (20× or 100× magnification).