PO and ID BCG vaccination in humans induce distinct mucosal and systemic immune responses and CD4+ T cell transcriptomal molecular signatures.

Protective efficacy of Bacillus Calmette-Guérin (BCG) may be affected by the methods and routes of vaccine administration. We have studied the safety and immunogenicity of oral (PO) and/or intradermal (ID) administration of BCG in healthy human subjects. No major safety concerns were detected in the 68 healthy adults vaccinated with PO and/or ID BCG. Although both PO and ID BCG could induce systemic Th1 responses capable of IFN-γ production, ID BCG more strongly induced systemic Th1 responses. In contrast, stronger mucosal responses (TB-specific secretory IgA and bronchoalveolar lavage T cells) were induced by PO BCG vaccination. To generate preliminary data comparing the early gene signatures induced by mucosal and systemic BCG vaccination, CD4+ memory T cells were isolated from subsets of BCG vaccinated subjects pre- (Day 0) and post-vaccination (Days 7 and 56), rested or stimulated with BCG infected dendritic cells, and then studied by Illumina BeadArray transcriptomal analysis. Notably, distinct gene expression profiles were identified both on Day 7 and Day 56 comparing the PO and ID BCG vaccinated groups by GSEA analysis. Future correlation analyses between specific gene expression patterns and distinct mucosal and systemic immune responses induced will be highly informative for TB vaccine development.

Allelic mRNA expression imbalance in C-type lectins reveals a frequent regulatory SNP in the human surfactant protein A (SP-A) gene.

Genetic variation in C-type lectins influences infectious disease susceptibility but remains poorly understood. We used allelic mRNA expression imbalance (AEI) technology for surfactant protein (SP)-A1, SP-A2, SP-D, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), macrophage mannose receptor (MRC1) and Dectin-1, expressed in human macrophages and/or lung tissues. Frequent AEI, an indicator of regulatory polymorphisms, was observed in SP-A2, SP-D and DC-SIGN. AEI was measured for SP-A2 in 38 lung tissues using four marker single-nucleotide polymorphisms (SNPs) and was confirmed by next-generation sequencing of one lung RNA sample. Genomic DNA at the SP-A2 DNA locus was sequenced by Ion Torrent technology in 16 samples. Correlation analysis of genotypes with AEI identified a haplotype block, and, specifically, the intronic SNP rs1650232 (30% minor allele frequency); the only variant consistently associated with an approximately twofold change in mRNA allelic expression. Previously shown to alter a NAGNAG splice acceptor site with likely effects on SP-A2 expression, rs1650232 generates an alternative splice variant with three additional bases at the start of exon 3. Validated as a regulatory variant, rs1650232 is in partial linkage disequilibrium with known SP-A2 marker SNPs previously associated with risk for respiratory diseases including tuberculosis. Applying functional DNA variants in clinical association studies, rather than marker SNPs, will advance our understanding of genetic susceptibility to infectious diseases.

Role of C-type lectins in mycobacterial infections.

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.

Pulmonary surfactant in innate immunity and the pathogenesis of tuberculosis.

Components of the innate immune system serve to protect the host from invading pathogens prior to the generation of a directed immune response, and influence the manner in which the directed immune response develops. The pulmonary surfactant system consists of a complex array of proteins and lipids that reduce surface tension of the alveoli, and appears to play an essential role in innate immunity. Investigators have recently gained insight into the interactions between components of the surfactant system and the respiratory pathogen Mycobacterium tuberculosis. It is likely that pulmonary surfactant and other innate immune determinants play significant roles in the pathogenesis of tuberculosis.

Gallium disrupts iron metabolism of mycobacteria residing within human macrophages.

Mycobacterium tuberculosis and M. avium complex (MAC) enter and multiply within monocytes and macrophages in phagosomes. In vitro growth studies using standard culture media indicate that siderophore-mediated iron (Fe) acquisition plays a critical role in the growth and metabolism of both M. tuberculosis and MAC. However, the applicability of such studies to conditions within the macrophage phagosome is unclear, due in part to the absence of experimental means to inhibit such a process. Based on the ability of gallium (Ga(3+)) to concentrate within mononuclear phagocytes and on evidence that Ga disrupts cellular Fe-dependent metabolic pathways by substituting for Fe(3+) and failing to undergo redox cycling, we hypothesized that Ga could disrupt Fe acquisition and Fe-dependent metabolic pathways of mycobacteria. We find that Ga(NO(3))(3) and Ga-transferrin produce an Fe-reversible concentration-dependent growth inhibition of M. tuberculosis strains and MAC grown extracellularly and within human macrophages. Ga is bactericidal for M. tuberculosis growing extracellularly and within macrophages. Finally, we provide evidence that exogenously added Fe is acquired by intraphagosomal M. tuberculosis and that Ga inhibits this Fe acquisition. Thus, Ga(NO(3))(3) disruption of mycobacterial Fe metabolism may serve as an experimental means to study the mechanism of Fe acquisition by intracellular mycobacteria and the role of Fe in intracellular survival. Furthermore, given the inability of biological systems to discriminate between Ga and Fe, this approach could have broad applicability to the study of Fe metabolism of other intracellular pathogens.

Virulent strains of Helicobacter pylori demonstrate delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages.

Helicobacter pylori colonizes the gastric epithelium of approximately 50% of the world's population and plays a causative role in the development of gastric and duodenal ulcers. H. pylori is phagocytosed by mononuclear phagocytes, but the internalized bacteria are not killed and the reasons for this host defense defect are unclear. We now show using immunofluorescence and electron microscopy that H. pylori employs an unusual mechanism to avoid phagocytic killing: delayed entry followed by homotypic phagosome fusion. Unopsonized type I H. pylori bound readily to macrophages and were internalized into actin-rich phagosomes after a lag of approximately 4 min. Although early (10 min) phagosomes contained single bacilli, H. pylori phagosomes coalesced over the next approximately 2 h. The resulting "megasomes" contained multiple viable organisms and were stable for 24 h. Phagosome-phagosome fusion required bacterial protein synthesis and intact host microtubules, and both chloramphenicol and nocodazole increased killing of intracellular H. pylori. Type II strains of H. pylori are less virulent and lack the cag pathogenicity island. In contrast to type I strains, type II H. pylori were rapidly ingested and killed by macrophages and did not stimulate megasome formation. Collectively, our data suggest that megasome formation is an important feature of H. pylori pathogenesis.

C1q-containing immune complexes purified from sera of juvenile rheumatoid arthritis patients mediate IL-8 production by human synoviocytes: role of C1q receptors.

Immune complexes that vary in size and composition are present in the sera and synovial fluid of juvenile rheumatoid arthritis (JRA) patients. They are believed to be potent inducers of the ongoing inflammatory process in JRA. However, the precise composition and role of these complexes in the pathophysiology of JRA remain unclear. We hypothesized that circulating ICs have the potential to interact with resident joint synovial fibroblasts (synoviocytes) and induce the expression of inflammatory cytokines. To test this hypothesis, cultures of synoviocytes from healthy individuals were treated with ICs isolated from the sera of JRA patients. Studies reported in this work demonstrate that IgM affinity-purified ICs from the sera of JRA patients contain IgM, C1q, IgG, and C3 to a variable extent. These ICs induce IL-8 mRNA and protein production in normal synoviocytes. Our data indicate that C1q in these ICs mediates, in part, IL-8 induction in synoviocytes. This is based on our findings of C1q-binding proteins for collagen stalks (cC1qR) and globular heads (gC1q-binding protein) of C1q in synoviocytes. In addition, collagen stalk and to some extent globular head fragments of C1q inhibit IC-mediated IL-8 induction in synoviocytes. Together, these findings provide evidence for a novel mechanism of IL-8 production by synoviocytes, which could play a key role in inflammation by recruiting leukocytes to synovial tissue and fluid-and subsequently contributing to joint disease.

Surfactant protein D binds to Mycobacterium tuberculosis bacilli and lipoarabinomannan via carbohydrate-lectin interactions resulting in reduced phagocytosis of the bacteria by macrophages.

Surfactant protein-D (SP-D) is a collectin produced in the distal lung airspaces that is believed to play an important role in innate pulmonary immunity. Naive immunologic responses to Mycobacterium tuberculosis (M.tb) are especially important in the lung, since entry of this inhaled pathogen into the alveolar macrophage is a pivotal event in disease pathogenesis. Here we investigated SP-D binding to M.tb and the effect of this binding on the adherence of M. tb to human macrophages. These studies demonstrate specific binding of SP-D to M.tb that is saturable, calcium dependent, and carbohydrate inhibitable. In addition to purified SP-D, SP-D in bronchoalveolar lavage fluids from healthy donors and patients with alveolar proteinosis also binds to M.tb. Incubation of M.tb with SP-D results in agglutination of the bacteria. In contrast to its binding to M.tb, SP-D binds minimally to the avirulent Mycobacterium smegmatis. SP-D binds predominantly to lipoarabinomannan from the virulent Erdman strain of M.tb, but not the lipoarabinomannan from M. smegmatis. The binding of SP-D to Erdman lipoarabinomannan is mediated by the terminal mannosyl oligosaccharides of this lipoglycan. Incubation of M.tb with subagglutinating concentrations of SP-D leads to reduced adherence of the bacteria to macrophages (62.7% of control adherence +/- 3.3% SEM, n = 8), whereas incubation of bacteria with surfactant protein A leads to significantly increased adherence to monocyte-derived macrophages. These data provide evidence for specific binding of SP-D to M. tuberculosis and indicate that SP-D and surfactant protein A serve different roles in the innate host response to this pathogen in the lung.

Characterization of mannose receptor-dependent phagocytosis mediated by Mycobacterium tuberculosis lipoarabinomannan.

The macrophage mannose receptor (MR) along with complement receptors mediates phagocytosis of the M. tuberculosis virulent strains Erdman and H37Rv. We have determined that the terminal mannosyl units of the M. tuberculosis surface lipoglycan, lipoarabinomannan (LAM), from the Erdman strain serve as ligands for the MR. The biology of the MR (receptor binding and trafficking) in response to phagocytic stimuli is not well characterized. This study analyzes the MR-dependent phagocytosis mediated by Erdman LAM presented on a 1-micron-diameter phagocytic particle. Erdman LAM microspheres exhibited a time- and dose-dependent rapid increase in attachment and internalization by human monocyte-derived macrophages (MDMs). In contrast, internalization of LAM microspheres by monocytes was minimal. Microsphere internalization by MDMs was visualized and quantitated by immunofluorescence and confocal and electron microscopy and resembled conventional phagocytosis. Phagocytosis of LAM microspheres by MDMs was energy, cytoskeleton, and calcium dependent and was mannan inhibitable. Trypsin treatment of MDMs at 37 degrees C, which depleted surface and recycling intracellular pools of the MR, reduced the subsequent attachment of LAM microspheres. Trypsin treatment at 4 degrees C allowed for subsequent recovery of LAM microsphere phagocytosis at 37 degrees C by recycled MRs. Pretreatment of MDMs with cycloheximide influenced LAM microsphere phagocytosis to only a small extent, indicating that MR-dependent phagocytosis of the microspheres was occurring primarily by preformed recycled receptors. This study characterizes the requirements for macrophage phagocytosis of a LAM-coated particle mediated by the MR. This model will be useful in further characterization of the intracellular pathway taken by phagocytic particles coated with different LAM types in macrophages following ingestion.

Mycobacterium tuberculosis and the complement system.

Accumulated evidence to date confirms the importance of the C3-CR pathway in the phagocytosis of pathogenic mycobacteria. Detailed receptor-ligand studies for phagocytosis are creating the framework to test the hypothesis that the entry pathway for these bacteria influences the immediate host cell response and their intracellular fate. These types of study are particularly important for improving our understanding of the outcome of primary infection in humans, where the number of bacilli is presumed to be very low.

Differences in mannose receptor-mediated uptake of lipoarabinomannan from virulent and attenuated strains of Mycobacterium tuberculosis by human macrophages.

Phagocytosis of the virulent Erdman and H37Rv strains of Mycobacterium tuberculosis, but not that of the attenuated H37Ra strain, by human macrophages is mediated by the mannose receptor (MR) in addition to complement receptors. We have recently determined that a major capsular lipoglycan, lipoarabinomannan (LAM), from the Erdman strain serves as a ligand for the MR during phagocytosis of bacteria. In this study we directly compare uptake of Erdman, H37Rv, and H37Ra LAM by human macrophages and assess the relative contribution of the MR in this process. Microspheres coated with LAM served as model phagocytic particles for studies of LAM as a capsular ligand. Uptake (37 degrees C) of LAM microspheres by monocyte-derived macrophages was greatest for Erdman LAM and intermediate for H37Rv and H37Ra LAM compared with that of buffer microspheres or microspheres coated with LAM from a nontuberculosis strain of mycobacterium (AraLAM). Inhibition of microsphere uptake in the presence of mannan or mannose-BSA was highest for Erdman LAM (75 +/- 8 and 50 +/- 7%, respectively) and H37Rv LAM (57 +/- 13 and 21 +/- 5%, respectively) relative to H37Ra LAM (36 +/- 16 and 22 +/- 11 %, respectively). Inhibition of microsphere uptake in the presence of anti-MR Ab followed a similar pattern: Erdman LAM (80 +/- 9%) > H37Rv LAM (53 +/- 1%) > H37Ra LAM (26 +/- 12%). Attachment (4 degrees C) of microspheres coated with Erdman LAM, H37Rv LAM, and H37Ra LAM was enhanced 12-, 5-, and 4-fold, respectively, compared with that of microspheres coated with AraLAM, and mannose-BSA inhibited attachment of these microspheres by 82 +/- 7, 69 +/- 8, and 12 +/- 17%. Galactose-BSA did not inhibit attachment of any LAM microsphere groups. Chromatographic analyses of mild acid hydrolysates of LAM from Erdman, H37Rv, and H37Ra all revealed the major terminal dimannosyl units. These studies demonstrate differences in the ability of LAM from different M. tuberculosis strains to mediate adherence to macrophages and to serve as ligands for the macrophage MR despite the presence of terminal dimannosyl units. Thus, these studies point toward other subtle structural alterations in LAM among strains that influence initial interactions with human phagocytes.

Activation of phospholipase D is tightly coupled to the phagocytosis of Mycobacterium tuberculosis or opsonized zymosan by human macrophages.

Phagocytosis of Mycobacterium tuberculosis by human mononuclear phagocytes is mediated primarily by complement receptors (CRs) but the transmembrane signaling mechanisms that regulate phagocytosis of the bacterium are unknown. We have analyzed the activation of phospholipase D (PLD) during phagocytosis of the virulent Erdman and attenuated H37Ra strains of M. tuberculosis by human monocyte-derived macrophages (MDMs), radiolabeled with [3H]-lyso-phosphatidylcholine. Phagocytosis of either Erdman or H37Ra M. tuberculosis in the presence of autologous non-immune serum was associated with a 2.5-3-fold increase in phosphatidic acid (PA). Definitive evidence for activation of PLD by M. tuberculosis was provided by markedly increased generation of the PLD-specific product phosphatidylethanol (PEt) (9.9-fold increases in [3H]-PEt for both Erdman and H37Ra strains compared to control, P < 0.001, n = 12), in the presence of 0.5% ethanol. Phagocytosis of opsonized zymosan (OZ), which is also mediated by CRs, was similarly associated with activation of PLD (12.2-fold increase in PEt, P < 0.001, n = 12). The competitive PLD inhibitor 2,3-diphosphoglycerate (2,3-DPG) produced concentration-dependent inhibition of PLD activity stimulated by either M. tuberculosis (-78 +/- 8%) or OZ (-73 +/- 6%). Inhibition of PLD by 2,3-DPG was associated with concentration-dependent reductions in phagocytosis of M. tuberculosis (-74 +/- 4%) and OZ (-68 +/- 5%). Addition of purified PLD from Streptomyces chromofuscus to 2,3-DPG-treated macrophages restored phagocytosis of M. tuberculosis to control levels. Inhibition of M. tuberculosis- or OZ-stimulated PA generation by ethanol was associated with concentration-dependent reductions in phagocytosis of both particles. Incubation of MDMs with either Erdman or H37Ra M. tuberculosis, or OZ, resulted in rapid (onset 1 min) and sustained (60 min) increases in the tyrosine phosphorylation (Tyr-P) of multiple MDM proteins. Prominent Tyr-P was noted in proteins of 150, 95, 72, 56, and 42 kD. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A reduced M. tuberculosis-stimulated PLD activity by 66-84%. Inhibition of PLD activity by genistein or herbimycin A was associated with inhibition of phagocytosis of M. tuberculosis and OZ. These data demonstrate that PLD is activated during macrophage phagocytosis of M. tuberculosis or OZ, that PTKs are involved in this stimulation of PLD, and that the extent of phagocytosis of these particles is tightly coupled to activation of PLD.

Pulmonary surfactant protein A mediates enhanced phagocytosis of Mycobacterium tuberculosis by a direct interaction with human macrophages.

During initial infection with Mycobacterium tuberculosis, bacteria that reach the distal airspaces of the lung are phagocytosed by alveolar macrophages in the presence of pulmonary surfactant. Here we have examined the role of surfactant-associated protein A (SP-A) in phagocytosis of the virulent Erdman strain of M. tuberculosis by human monocyte-derived macrophages (MDMs) and human alveolar macrophages (HAMs). Macrophage monolayers incubated with soluble SP-A from alveolar proteinosis patients (APP SP-A4) and recombinant rat SP-A (SP-Ahyp) demonstrated enhanced adherence of M. tuberculosis, 82 +/- 17% and 49 +/- 18%, respectively. Removal of SP-A from monolayers by washing before adding bacteria did not diminish the enhanced adherence. Fluorescence microscopy demonstrated that washed monolayers contained intracellular rather than surface-bound SP-A. These studies indicated a direct interaction between SP-A and the macrophage in mediating enhanced adherence of M. tuberculosis. Consistent with this interpretation, macrophage monolayers formed on human or rat SP-A (substrate SP-A) demonstrated enhanced adherence of M. tuberculosis to their apical surface (APP SP-A and native rat SP-A increased M. tuberculosis adherence by 102 +/- 16% and 102 +/- 25%, respectively). Electron microscopy demonstrated increased numbers of phagocytosed bacteria in APP SP-A-treated MDM cross-sections. SP-A proteins devoid of carbohydrate failed to enhance M. tuberculosis adherence to macrophages. In contrast, heat-denatured APP SP-A enhanced adherence of bacteria equivalent to that of intact glycoprotein. Thus, the carbohydrate moieties of SP-A appear to be critical in the SP-A-macrophage interaction. Finally, mannan and anti-mannose receptor Ab completely inhibited the enhanced phagocytosis of M. tuberculosis observed with APP SP-A, providing evidence for up-regulation of macrophage mannose receptor activity. These studies implicate SP-A as an important modulator of alveolar macrophage function that results in an enhanced potential for M. tuberculosis to gain access to its intracellular niche.

Binding of the terminal mannosyl units of lipoarabinomannan from a virulent strain of Mycobacterium tuberculosis to human macrophages.

Recent studies from this laboratory have demonstrated that macrophage phagocytosis of virulent strains (Erdman and H37Rv), but not the attenuated H37Ra strain of Mycobacterium tuberculosis, is mediated by phagocyte mannose receptors (MR) in addition to complement receptors (CR1 and the leukocyte integrins CR3 and CR4). Lipoarabinomannan (LAM) is a major surface lipoglycan of M. tuberculosis. LAM from the Erdman strain (Man-LAM) contains mannose oligosaccharides at the terminal portions of the molecule. This study investigated the ability of ManLAM to serve as a microbial ligand in adherence to human monocyte-derived macrophages (MDM). Polystyrene microspheres were coated with known amounts of purified ManLAM, LAM without the terminal mannosyl units from an avirulent mycobacterium (AraLAM), lipomannan (LM), or buffer and incubated with MDM monolayers in the absence of serum. The presence of LAM on microspheres was confirmed by indirect immunofluorescence studies. Microspheres coated with ManLAM demonstrated a more than threefold increase in adherence to MDM when compared with microspheres coated with AraLAM, LM, or buffer and the low levels of adherence of microspheres in the latter three groups were comparable. Compared with control monolayers, selective down-modulation of MDM MR on a mannan substrate abrogated the enhanced adherence of microspheres mediated by ManLAM. Adherence of microspheres coated with AraLAM, LM, or buffer was not influenced by MR modulation. To confirm the importance of the terminal mannosyl units of ManLAM in the enhanced adherence of ManLAM microspheres to MDM, these units were selectively removed by exomannosidase treatment. The structure of LAM products before and after enzyme treatment was confirmed by high performance anion exchange chromatography with pulsed amperometric detection. Removal of the terminal mannosyl units abolished the capacity of ManLAM to mediate enhanced adherence of microspheres to MDM. Finally, preincubation of Erdman M. tuberculosis with CS-40, a mAb directed against LAM, resulted in a consistent inhibition of adherence of the bacteria to MDM (up to 49% inhibition), confirming a role for ManLAM on intact bacteria in adherence to MDM. Thus, we provide evidence for a novel receptor-ligand pathway in phagocytosis of M. tuberculosis that consists of MR on macrophages and mannosyl units at the terminal end of ManLAM, a major microbial surface lipoglycan.