Publications

Worldwide clinical cases due to multi drug- and extensively drug-resistant strains of Mycobacterium tuberculosis (M.tb) are increasing making the need for new therapies more critical than ever. A major obstacle for designing new drugs to treat mycobacterial infections is our limited knowledge of the interface between the bacillus (especially M.tb) and its host. The pulmonary innate immune system plays a key role in the recognition of microbes entering via the respiratory route. Although the specificity of this system is broad and based on the recognition of pathogen-associated molecular patterns (PAMPs), it is uniquely regulated to limit inflammation and thereby prevent damage to the gas-exchanging alveoli. Pulmonary surfactant proteins A and D (SP-A and SP-D) are collagenous, soluble, C-type (Ca(2+)-dependent) lectins (named collectins) of the lung innate immune system that are secreted into the alveoli by resident type II alveolar epithelial cells and distal bronchiolar Clara cells. The related collectin in serum, mannose-binding lectin/protein (MBL or MBP), provides first-line defense against several microbes. Phagocytes represent the first cellular defense in the alveoli and their surface is rich in C-type lectin pattern recognition receptors (PRRs), including the mannose receptor (MR), dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) and DC-associated C-type lectin-1 (Dectin-1). This review will discuss the important roles of the cell-associated C-type lectin PRRs and soluble collectins in the innate immune response to mycobacterial infections, and will present the current state of knowledge regarding the potential uses of these C-type lectins in therapy against infections, focusing on M.tb.

Iron (Fe) acquisition is essential for the growth of intracellular Mycobacterium tuberculosis (M.tb). How this occurs is poorly understood. Hereditary hemochromatosis is an inherited disease in which most cells become overloaded with Fe. However, hereditary hemochromatosis macrophages have lower than normal levels of intracellular Fe. This suggests M.tb growth should be slower in those cells if macrophage intracellular Fe is used by M.tb. Therefore, we compared trafficking and acquisition of transferrin (Tf)- and lactoferrin (Lf)-chelated Fe by M.tb within the phagosome of monocyte-derived macrophages (MDM) from healthy controls and subjects with hereditary hemochromatosis. M.tb in both sets of macrophages acquired more Fe from Lf than Tf. Fe acquisition by M.tb within hereditary hemochromatosis macrophages was decreased by 84% from Tf and 92% from Lf relative to that in healthy control macrophages. There was no difference in Fe acquired from Tf and Lf by the two macrophage phenotypes. Both acquired 3 times more Fe from Lf than Tf. M.tb infection and incubation with interferon gamma (IFN-gamma) reduced macrophage Fe acquisition by 20% and 50%, respectively. Both Tf and Lf colocalized with M.tb phagosomes to a similar extent, independent of macrophage phenotype. M.tb growth was 50% less in hereditary hemochromatosis macrophages. M.tb growing within macrophages from subjects with hereditary hemochromatosis acquire less Fe compared with healthy controls. This is associated with reduced growth of M.tb. These data support a role for macrophage intracellular Fe as a source for M.tb growth.

Hepcidin is an antimicrobial peptide produced by the liver in response to inflammatory stimuli and iron overload. Hepcidin regulates iron homeostasis by mediating the degradation of the iron export protein ferroportin 1, thereby inhibiting iron absorption from the small intestine and release of iron from macrophages. Here, we examined the expression of hepcidin in macrophages infected with the intracellular pathogens Mycobacterium avium and Mycobacterium tuberculosis. Stimulation of the mouse RAW264.7 macrophage cell line and mouse bone marrow-derived macrophages with mycobacteria and IFN-gamma synergistically induced high levels of hepcidin mRNA and protein. Similar results were obtained using the human THP-1 monocytic cell line. Stimulation of macrophages with the inflammatory cytokines IL-6 and IL-beta did not induce hepcidin mRNA expression. Iron loading inhibited hepcidin mRNA expression induced by IFN-gamma and M. avium, and iron chelation increased hepcidin mRNA expression. Intracellular protein levels and secretion of hepcidin were determined by a competitive chemiluminescence ELISA. Stimulation of RAW264.7 cells with IFN-gamma and M. tuberculosis induced intracellular expression and secretion of hepcidin. Furthermore, confocal microscopy analyses showed that hepcidin localized to the mycobacteria-containing phagosomes. As hepcidin has been shown to possess direct antimicrobial activity, we investigated its activity against M. tuberculosis. We found that hepcidin inhibited M. tuberculosis growth in vitro and caused structural damage to the mycobacteria. In summary, our data show for the first time that hepcidin localizes to the phagosome of infected, IFN-gamma-activated cells and has antimycobacterial activity.

AcpA of Francisella spp. is a respiratory-burst-inhibiting acid phosphatase that also exhibits phospholipase C activity. To better understand the molecular basis of AcpA in virulence, a deletion of acpA was constructed in Francisella novicida. The phosphatase and lipase activities were reduced 10-fold and 8-fold, respectively, in the acpA mutant compared to the wild type and were found mostly associated with the outer membrane. The acpA mutant was more susceptible to intracellular killing than the wild-type strain in the THP-1 human macrophage-like cell line. In addition, mice infected with the acpA mutant survived longer than the wild-type strain and were less fit than the wild-type strain in competition infection assays. Transmission electron microscopy showed that the acpA mutant was delayed in escape from macrophage phagosomes, as more than 75% of acpA mutant bacteria could still be found inside phagosomes after 12 h of infection in THP-1 cells and human monocyte-derived macrophages, whereas most of the wild-type bacteria had escaped from the phagosome by 6 h postinfection. Thus, AcpA affects intracellular trafficking and the fate of Francisella within host macrophages.

Francisella tularensis, the causative agent of tularemia, is phagocytosed by immune cells such as monocytes and macrophages. Instead of being destroyed in the phagolysosome, the bacterium escapes the phagosome and replicates within the host cytosol. Recent studies indicate that phagosomal escape may have a major impact on the nature of the inflammatory cytokine response to infection. To better understand the host cell response to Francisella infection, we exposed human peripheral blood monocytes to Francisella novicida and analyzed transcriptional changes using high-density oligonucleotide microarrays. Results showed a nearly 300-fold up-regulation of transcripts for the p19 subunit of IL-23, and a nearly 18-fold up-regulation for the p40 subunit of IL-12. IL-23 is formed by the heterodimerization of p19 and p40, and is an important cytokine of the innate immune response. Up-regulation of p19 and p40 was confirmed at the protein level by Western blotting and ELISA analyses, and was found to be largely dependent on PI3K and NF-kappaB activity. Studies using medium from infected monocytes with or without a p19 blocking Ab showed that the secreted IL-23 induced IFN-gamma production from NK cells, suggesting a potential biologically important role for IL-23 in host defense. Finally, infection of human monocytes by the highly virulent Francisella SCHU S4 strain likewise led to IL-23 production, suggesting that the IL-23 response may be relevant during tularemia.

Inhalational pneumonic tularemia, caused by Francisella tularensis, is lethal in humans. F. tularensis is phagocytosed by macrophages followed by escape from phagosomes into the cytoplasm. Little is known of the phagocytic mechanisms for Francisella, particularly as they relate to the lung and alveolar macrophages. Here we examined receptors on primary human monocytes and macrophages which mediate the phagocytosis and intracellular survival of F. novicida. F. novicida association with monocyte-derived macrophages (MDM) was greater than with monocytes. Bacteria were readily ingested, as shown by electron microscopy. Bacterial association was significantly increased in fresh serum and only partially decreased in heat-inactivated serum. A role for both complement receptor 3 (CR3) and Fcgamma receptors in uptake was supported by studies using a CR3-expressing cell line and by down-modulation of Fcgamma receptors on MDM, respectively. Consistent with Fcgamma receptor involvement, antibody in nonimmune human serum was detected on the surface of Francisella. In the absence of serum opsonins, competitive inhibition of mannose receptor (MR) activity on MDM with mannan decreased the association of F. novicida and opsonization of F. novicida with lung collectin surfactant protein A (SP-A) increased bacterial association and intracellular survival. This study demonstrates that human macrophages phagocytose more Francisella than monocytes with contributions from CR3, Fcgamma receptors, the MR, and SP-A present in lung alveoli.

The Mycobacterium tuberculosis (M.tb) envelope is highly mannosylated with phosphatidyl-myo-inositol mannosides (PIMs), lipomannan, and mannose-capped lipoarabinomannan (ManLAM). Little is known regarding the interaction between specific PIM types and host cell C-type lectin pattern recognition receptors. The macrophage mannose receptor (MR) and dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells engage ManLAM mannose caps and regulate several host responses. In this study, we analyzed the association of purified PIM families (f, separated by carbohydrate number) and individual PIM species (further separated by fatty acid number) from M.tb H(37)R(v) with human monocyte-derived macrophages (MDMs) and lectin-expressing cell lines using an established bead model. Higher-order PIMs preferentially associated with the MR as demonstrated by their reduced association with MDMs upon MR blockade and increased binding to COS-1-MR. In contrast, the lower-order PIM(2)f associated poorly with MDMs and did not bind to COS-1-MR. Triacylated PIM species were recognized by MDM lectins better than tetra-acylated species and the degree of acylation influenced higher-order PIM association with the MR. Moreover, only higher-order PIMs that bind the MR showed a significant increase in phagosome-lysosome fusion upon MR blockade. In contrast with the MR, the PIM(2)f and lipomannan were recognized by DC-SIGN comparable to higher-order PIMs and ManLAM, and the association was independent of their degree of acylation. Thus, recognition of M.tb PIMs by host cell C-type lectins is dependent on both the nature of the terminal carbohydrates and degree of acylation. Subtle structural differences among the PIMs impact host cell recognition and response and are predicted to influence the intracellular fate of M.tb.

A crucial step in infection is the initial attachment of a pathogen to host cells or tissue. Mycobacterium tuberculosis has evolved multiple strategies for establishing an infection within the host. The pulmonary microenvironment contains a complex milieu of pattern recognition molecules of the innate immune system that play a role in the primary response to inhaled pathogens. Encounters of M. tuberculosis with these recognition molecules likely influence the outcome of the bacillus-host interaction. Here we use a novel fluid shear assay to investigate the binding of M. tuberculosis to innate immune molecules that are produced by pulmonary epithelial cells and are thought to play a role in the lung innate immune response. Virulent and attenuated M. tuberculosis strains bound best to immobilized human fibronectin (FN) and surfactant protein A (SP-A) under this condition. Binding under fluid shear conditions was more consistent and significant compared to binding under static conditions. Soluble FN significantly increased the adherence of both virulent and attenuated M. tuberculosis strains to human primary small airway epithelial cells (SAEC) under fluid shear conditions. In contrast, SP-A and SP-D effects on bacterial adherence to SAEC differed between the two strains. The use of a fluid shear model to simulate physiological conditions within the lung and select for high-affinity binding interactions should prove useful for studies that investigate interactions between M. tuberculosis and host innate immune determinants.

In the noninflamed lung, surfactant protein A (SP-A) acts as an anti-inflammatory molecule through its effects on macrophage (MPhi) function, modulating cytokine and reactive oxygen and nitrogen intermediate production. The receptors responsible for these effects of SP-A on human MPhi are not clear, although SP-A binding to several proteins has been described. In this study, we demonstrate high-affinity specific binding of SP-A to primary human MPhi. SP-A binding was inhibited by EGTA, indicating calcium dependence. However, mannan did not inhibit SP-A binding, suggesting that binding is mediated by a direct protein-protein interaction that does not involve carbohydrate recognition. Our laboratory has previously shown that SP-A is rapidly endocytosed by human MPhi into discrete vesicles. Although previous work indicates that SP-A is ultimately degraded by murine MPhi over time, the trafficking pathway of SP-A through MPhi after uptake has not been reported and is of potential biological importance. We examined trafficking of SP-A in human MPhi by electron and confocal microscopy and show for the first time that SP-A is endocytosed by primary human MPhi through clathrin-coated pits and colocalizes sequentially over time with the early endosome marker EEA1, late endosome marker lamp-1, and lysosome marker cathepsin D. We conclude that SP-A binds to receptor(s) on human MPhi, is endocytosed by a receptor-mediated, clathrin-dependent process, and trafficks through the endolysosomal pathway. These studies provide further insight into the interactions of SP-A with the MPhi cell surface and intracellular compartments that play important roles in SP-A modulation of lung MPhi biology.