lactoferrin and Tuberculosis

There is great need for a therapeutic that would limit tuberculosis related pathology and thus curtail spread of disease between individuals by establishing a "firebreak" to slow transmission. A promising avenue to increase current therapeutic efficacy may be through incorporation of adjunct components that slow or stop development of aggressive destructive pulmonary pathology. Lactoferrin, an iron-binding glycoprotein found in mucosal secretions and granules of neutrophils, is just such a potential adjunct therapeutic agent. The focus of this review is to explore the utility of lactoferrin to serve as a therapeutic tool to investigate "disruption" of the mycobacterial granuloma. Proposed concepts for mechanisms underlying lactoferrin efficacy to control immunopathology are supported by data generated based on in vivo models using nonpathogenic trehalose 6,6'-dimycolate (TDM, cord factor).

Topics: Animals; Cord Factors; Granuloma; Humans; Lactoferrin; Mice; Tuberculosis

Host antimicrobial mechanisms reduce iron availability to pathogens. Iron proteins influencing the innate immune response include hepcidin, lactoferrin, siderocalin, haptoglobin, hemopexin, Nramp1, ferroportin and the transferrin receptor. Numerous global health threats are influenced by iron status and provide examples of our growing understanding of the connections between infection and iron metabolism.

Topics: Animals; Antimicrobial Cationic Peptides; Carrier Proteins; Cation Transport Proteins; Hepcidins; HIV; HIV Infections; Humans; Immunity, Innate; Iron; Lactoferrin; Lipocalin-2; Malaria; Mycobacterium tuberculosis; Plasmodium; Tuberculosis

Availability of iron is a key factor in the survival and multiplication of Mycobacterium tuberculosis (M.tb) within host macrophage phagosomes. Despite host cell iron regulatory machineries attempts to deny supply of this essential micronutrient, intraphagosomal M.tb continues to access extracellular iron. In the current study, we report that intracellular M.tb exploits mammalian secreted Glyceraldehyde 3-phosphate dehydrogenase (sGAPDH) for the delivery of host iron carrier proteins lactoferrin (Lf) and transferrin (Tf). Studying the trafficking of iron carriers in infected cells we observed that sGAPDH along with the iron carrier proteins are preferentially internalized into infected cells and trafficked to M.tb containing phagosomes where they are internalized by resident mycobacteria resulting in iron delivery. Collectively our findings provide a new mechanism of iron acquisition by M.tb involving the hijack of host sGAPDH. This may contribute to its successful pathogenesis and provide an option for targeted therapeutic intervention.

Topics: Animals; Biological Transport; Cell Line, Tumor; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Iron; L Cells; Lactoferrin; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Phagosomes; THP-1 Cells; Transferrin; Tuberculosis

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

Topics: Administration, Oral; Animals; Cord Factors; Cytokines; Female; Granuloma; Humans; Lactoferrin; Lung Diseases; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Recombinant Proteins; Tuberculosis

Lactoferrin (LF), a natural iron-binding protein, has previously demonstrated effectiveness in enhancing the Bacillus Calmette-Guérin (BCG) tuberculosis vaccine. This report investigates immune modulatory effects of Chinese hamster ovary (CHO) cell-expressed recombinant mouse and human LFs on mouse bone marrow-derived dendritic cells (BMDCs), comparing homologous and heterologous functions. BCG-infected BMDCs were cultured with LF, and examined for class II presentation molecule expression. Culturing of BCG-infected BMDCs with either LF decreased the class II molecule-expressing population. Mouse LF significantly increased the production of IL-12p40, IL-1β and IL-10, while human LF-treated BMDCs increased only IL-1β and IL-10. Overlaying naïve CD4 T-cells onto BCG-infected BMDCs cultured with mouse LF increased IFN-γ, whereas the human LF-exposed group increased IFN-γ and IL-17 from CD4 T cells. Overlay of naïve CD8 T cells onto BCG-infected BMDCs treated with mouse LF increased the production of IFN-γ and IL-17, while similar experiments using human LF only increased IL-17. This report is the first to examine mouse and human recombinant LFs in parallel experiments to assess murine DC function. These results detail the efficacy of the human LF counterpart used in a heterologous system to understand LF-mediated events that confer BCG efficacy against Mycobacterium tuberculosis challenge.

Topics: Animals; Antigen Presentation; Bacillus; CD4-Positive T-Lymphocytes; CHO Cells; Cricetinae; Cricetulus; Dendritic Cells; Gram-Positive Bacterial Infections; Humans; Interleukin-17; Lactoferrin; Mice; Mycobacterium tuberculosis; Recombinant Proteins; Tuberculosis

Differentiation of tuberculous granuloma (TG) from non-tuberculous granuloma (NG) is histopathologically difficult. We evaluated the usefulness of selected immunohistochemical markers to differentiate tuberculous granuloma (TG) and non-tuberculous granuloma (NG). We selected six biomarkers (FoxP3, TNF-beta, E-selectin [ESEL], indoleamine 2,3-dioxygenase [IDO], lactoferrin [LACT], and tartrate-resistant acid phosphatase [TRAP]) and immunohistochemically analyzed their expression in the presence of two types of granulomatous tissue samples, TG (n = 36) and NG (n = 31), using a microarray format. Three of those six biomarkers (LACT, IDO, and TNF-beta) were moderately accurate in discriminating TG from NG, individually and in combination, according to ROC analysis (AUC = 0.7-0.89, sensitivity = 55.6-77.8%, specificity = 71.0-100%). Our data indicate that selected immunohistochemical markers (LACT, IDO, and TNF-beta) can be used in ancillary tests to differentiate TG from NG in tissue samples. Further large-scale studies are required to validate our results.

Topics: Adolescent; Adult; Aged; Area Under Curve; Biomarkers; Child, Preschool; Diagnosis, Differential; Feasibility Studies; Female; Fixatives; Formaldehyde; Granuloma; Humans; Immunohistochemistry; Indoleamine-Pyrrole 2,3,-Dioxygenase; Lactoferrin; Lymphotoxin-alpha; Male; Middle Aged; Paraffin Embedding; Predictive Value of Tests; Retrospective Studies; ROC Curve; Tissue Array Analysis; Tissue Fixation; Tuberculosis; Young Adult

The pathophysiology of Mycobacterium tuberculosis (M.tb) infection is linked to the ability of the organism to grow within macrophages. Lung myeloid dendritic cells are a newly recognized reservoir of M.tb during infection. Iron (Fe) acquisition is critical for M.tb growth. In vivo, extracellular Fe is chelated to transferrin (TF) and lactoferrin (LF). We previously reported that M.tb replicating in human monocyte-dervied macrophages (MDM) can acquire Fe bound to TF, LF, and citrate, as well as from the MDM cytoplasm. Access of M.tb to Fe may influence its growth in macrophages and dendritic cells. In the present work we confirmed the ability of different strains of M.tb to grow in human myeloid dendritic cells in vitro. Fe acquired by M.tb replicating within dendritic cells from externally added Fe chelates varied with the Fe chelate present in the external media: Fe-citrate > Fe-LF > Fe-TF. Fe acquisition rates from each chelate did not vary over 7 days. M.tb within dendritic cells also acquired Fe from the dendritic cell cytoplasm, with the efficiency of Fe acquisition greater from cytoplasmic Fe sources, regardless of the initial Fe chelate from which that cytoplasmic Fe was derived. Growth and Fe acquisition results with human MDM were similar to those with dendritic cells. M.tb grow and replicate within myeloid dendritic cells in vitro. Fe metabolism of M.tb growing in either MDM or dendritic cells in vitro is influenced by the nature of Fe available and the organism appears to preferentially access cytoplasmic rather than extracellular Fe sources. Whether these in vitro data extend to in vivo conditions should be examined in future studies.

Topics: Biological Transport; Cells, Cultured; Dendritic Cells; Humans; Iron; Lactoferrin; Macrophages; Mycobacterium tuberculosis; Transferrin; Tuberculosis

Iron is an essential cofactor for both mycobacterial growth during infection and for a successful protective immune response by the host. The immune response partly depends on the regulation of iron by the host, including the tight control of expression of the iron-storage protein, ferritin. BCG vaccination can protect against disease following Mycobacterium tuberculosis infection, but the mechanisms of protection remain unclear. To further explore these mechanisms, splenocytes from BCG-vaccinated guinea pigs were stimulated ex vivo with purified protein derivative from M. tuberculosis and a significant down-regulation of ferritin light- and heavy-chain was measured by reverse-transcription quantitative-PCR (P≤0.05 and ≤0.01, respectively). The mechanisms of this down-regulation were shown to involve TNFα and nitric oxide. A more in depth analysis of the mRNA expression profiles, including genes involved in iron metabolism, was performed using a guinea pig specific immunological microarray following ex vivo infection with M. tuberculosis of splenocytes from BCG-vaccinated and naïve guinea pigs. M. tuberculosis infection induced a pro-inflammatory response in splenocytes from both groups, resulting in down-regulation of ferritin (P≤0.05). In addition, lactoferrin (P≤0.002), transferrin receptor (P≤0.05) and solute carrier family 11A1 (P≤0.05), were only significantly down-regulated after infection of the splenocytes from BCG-vaccinated animals. The results show that expression of iron-metabolism genes is tightly regulated as part of the host response to M. tuberculosis infection and that BCG-vaccination enhances the ability of the host to mount an iron-restriction response which may in turn help to combat invasion by mycobacteria.

Topics: Animals; BCG Vaccine; Cation Transport Proteins; Ferritins; Gene Expression Profiling; Guinea Pigs; Iron; Lactoferrin; Microarray Analysis; Mycobacterium tuberculosis; Real-Time Polymerase Chain Reaction; Receptors, Transferrin; Tuberculosis

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.

Topics: Animals; Cord Factors; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Granuloma; Interleukin-10; Lactoferrin; Lung; Lung Diseases; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Protein Biosynthesis; Transforming Growth Factor beta; Tuberculosis

Lactoferrin, an 80-kDa iron-binding protein with immune modulating properties, is a unique adjuvant component able to enhance efficacy of the existing Mycobacterium bovis Bacillus Calmette Guerin (BCG) vaccine to protect against murine model of tuberculosis. Although identified as having effects on macrophage presentation events, lactoferrin's capability to modulate dendritic cells (DCs) function when loaded with BCG antigens has not been previously recognized. In this study, the potential of lactoferrin to modulate surface expression of MHC II, CD80, CD86 and CD40 from bone marrow-derived dendritic cells (BMDCs) was examined. Generally, lactoferrin decreased pro-inflammatory cytokines [tumor necrosis factor (TNF)-alpha, IL-6 and IL-12p40] and chemokines [macrophage inflammatory protein (MIP)-1alpha and MIP-2] and increased regulatory cytokine, transforming growth factor-beta1 and a T-cell chemotatic factor, monocyte chemotactic protein-1, from uninfected or BCG-infected BMDCs. Culturing BCG-infected BMDCs with lactoferrin also enhanced their ability to respond to IFN-gamma activation through up-regulation of maturation markers: MHC I, MHC II and the ratio of CD86:CD80 surface expression. Furthermore, lactoferrin-exposed BCG-infected DCs increased stimulation of BCG-specific CD3(+)CD4(+) splenocytes, as defined by increasing IFN-gamma production. Finally, BCG-/lactoferrin-vaccinated mice possessed an increased pool of BCG antigen-specific IFN-gamma producing CD3(+)CD4(+)CD62L(-) splenocytes. These studies suggest a mechanism in which lactoferrin may exert adjuvant activity by enhancing DC function to promote generation of antigen-specific T cells.

Topics: Adjuvants, Immunologic; Animals; B7-1 Antigen; B7-2 Antigen; BCG Vaccine; CD4-Positive T-Lymphocytes; CD40 Antigens; CD8-Positive T-Lymphocytes; Cytokines; Dendritic Cells; Disease Models, Animal; Female; Histocompatibility Antigens Class II; Lactoferrin; Mice; Mice, Inbred C57BL; Mycobacterium bovis; Tuberculosis

The maintenance and multiplication of Mycobacteria tuberculosis (TB) and many other species of parasitic pathogen are dependent to varying, largely unidentified degrees upon a source of free iron within the host tissues. To combat these infections, the mammalian biosystem expresses an iron binding exocrine protein, lactoferrin, which scavenges and competes for free iron, thereby starving the parasite of its vital iron supply. TB mycobacteria are naturally endemic in the external environment, and once a latent, low level TB infection is established within the host tissues, a full blown proliferation of the mycobacteria population can be activated as soon as the levels of free iron are elevated within the host tissues. The increase in iron can be induced by several environmental and/or eco-genetic prerequisites that operate either singly or in a synergistic combination; factors such as iron rich water/foods, increased iron uptake/retention in the host tissues or an environmental/genetic induced reduction in the turn over of iron binding lactoferrin mediated immune defence against TB. Susceptibility to the full blown proliferation of TB pathogenesis is markedly increased as a result. This paper proposes that the recent dramatic increase in the incidence of bovine/badger TB across the UK can be correlated to the overall increase in acidification of the agricultural ecosystem, which, in turn, has induced a substantial elevation of soluble iron within the farm foodchain, thereby exacerbating susceptibility to TB infection within any mammalian species that is dependent upon these high iron ecosystems. The problem is further compounded by the routine use of 'anti-lactoferrin' levamisole based cattle wormers, which 'sensitise' the levamisole's target receptors, thereby down regulating the secretion of the iron binding lactoferrin molecule, which causes a reduction in the host's main line of defence against TB infection.

Topics: Animals; Cattle; Disease Susceptibility; Down-Regulation; Environment; Iron; Lactoferrin; Levamisole; Mustelidae; Mycobacterium tuberculosis; Protein Binding; Receptors, Cell Surface; Tuberculosis

The ability to control intracellular Mycobacterium tuberculosis (MTB) infection relies on cellular immunity and generation of a strong T-cell helper 1 (T(H)1) response. Lactoferrin, an iron-binding protein with immune regulatory functions, was investigated as an adjuvant to boost Mycobacterium bovis Bacillus Calmette-Guerin (BCG) efficacy. Lactoferrin was initially shown to augment IL-12(p40) production from macrophages stimulated with LPS. A single immunization of mice with Lactoferrin as an adjunct adjuvant resulted in amplified splenocyte proliferative response to heat-killed BCG, and elevated IL-12(p40) production with increased relative ratios of IL-12/IL-10. Furthermore, splenocyte recall response to HK-BCG was augmented for proinflammatory mediators, TNF-alpha, IL-1beta, and IL-6, approaching responses generated to complete Freund's adjuvant (CFA) immunized controls. Specific responses were identified, with significant elevation of IFN-gamma generated during antigenic recall. Subsequent aerosol challenge of Lactoferrin adjuvant immunized mice with virulent M. tuberculosis revealed decreased mycobacterial loads in the lung, and limitation of organism dissemination to a peripheral organ (spleen). These studies indicate that Lactoferrin can act as an adjunct adjuvant to augment cellular immunity and boost BCG efficacy for protection against subsequent challenge with virulent MTB.

Topics: Animals; BCG Vaccine; Cell Line, Tumor; Cytokines; Female; Interferon-gamma; Interleukin-12; Lactoferrin; Lipopolysaccharides; Lung; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium bovis; Mycobacterium tuberculosis; Spleen; T-Lymphocytes, Helper-Inducer; Th1 Cells; Tuberculosis; Virulence

We have reported that Mycobacterium tuberculosis residing within the phagosomes of human monocyte-derived macrophages (MDM) can acquire Fe from extracellular transferrin (TF) and sources within the MDM. In the lung, Fe is also bound to lactoferrin (LF) and low-molecular-weight chelates. We therefore investigated the ability of intraphagosomal M. tuberculosis to acquire Fe from these sources. M. tuberculosis acquired 30-fold and 3-fold more Fe from LF and citrate, respectively, compared to TF, in spite of similar MDM-associated Fe. M. tuberculosis infection decreased MDM-associated Fe relative to uninfected MDM as follows: TF (38.7%), citrate (21.1%), and LF (15.3%). M. tuberculosis Fe acquisition from extracellular chelates (exogenous source) and from endogenous MDM Fe initially acquired from the three chelates (endogenous source) was compared. M. tuberculosis Fe acquisition was similar from exogenous and endogenous sources supplied as Fe-TF. In contrast, there was much greater intracellular M. tuberculosis Fe uptake from LF and citrate from the exogenous than endogenous source. Gamma interferon (IFN-gamma) reduced MDM Fe uptake from each chelate by approximately 50% and augmented the M. tuberculosis-induced decrease in MDM Fe uptake from exogenous TF, but not from LF or citrate. IFN-gamma minimally decreased intracellular M. tuberculosis Fe acquisition from exogenous Fe-TF but significantly increased Fe uptake from LF and citrate. Intraphagosomal M. tuberculosis Fe acquisition from both exogenous and endogenous MDM sources, and the effect of IFN-gamma on this process, is influenced by the nature of the extracellular Fe chelate. M. tuberculosis has developed efficient mechanisms of acquiring Fe from a variety of Fe chelates that it likely encounters within the human lung.

Topics: Cells, Cultured; Citrates; Humans; Interferon-gamma; Iron; Lactoferrin; Macrophages; Mycobacterium tuberculosis; Phagocytosis; Phagosomes; Tuberculosis

As a resident of early endosomal phagosomes, Mycobacterium tuberculosis is connected to the iron uptake system of the host macrophage. beta-2-microglobulin (beta2m) knockout (KO) mice are more susceptible to tuberculosis than wild-type mice, which is generally taken as a proof for the role of major histocompatibility complex class I (MHC-I)-restricted CD8 T cells in protection against M. tuberculosis. However, beta2m associates with a number of MHC-I-like proteins, including HFE. This protein regulates transferrin receptor mediated iron uptake and mutations in its gene cause hereditary iron overload (hemochromatosis). Accordingly, beta2m-deficient mice suffer from tissue iron overload. Here, we show that modulating the extracellular iron pool in beta2m-KO mice by lactoferrin treatment significantly reduces the burden of M. tuberculosis to numbers comparable to those observed in MHC class I-KO mice. In parallel, the generation of nitric oxide impaired in beta2m-KO mice was rescued. Conversely, iron overload in the immunocompetent host exacerbated disease. Consistent with this, iron deprivation in infected resting macrophages was detrimental for intracellular mycobacteria. Our data establish: (a) defective iron metabolism explains the increased susceptibility of beta2m-KO mice over MHC-I-KO mice, and (b) iron overload represents an exacerbating cofactor for tuberculosis.

Topics: Animals; beta 2-Microglobulin; Disease Susceptibility; Hemochromatosis Protein; Histocompatibility Antigens Class I; Interferon-gamma; Iron Overload; Lactoferrin; Macrophages; Membrane Proteins; Mice; Mice, Knockout; Mycobacterium tuberculosis; Nitric Oxide; Receptors, Transferrin; Tuberculosis