Publications

Human alveolar macrophages (HAM) are primary bacterial niche and immune response cells during Mycobacterium tuberculosis (M.tb) infection, and human blood monocyte-derived macrophages (MDM) are a model for investigating M.tb-macrophage interactions. Here, we use a targeted RNA-Seq method to measure transcriptome-wide changes in RNA expression patterns of freshly obtained HAM (used within 6 h) and 6 day cultured MDM upon M.tb infection over time (2, 24 and 72 h), in both uninfected and infected cells from three donors each. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) uses primers targeting 18,574 mRNAs and 2,228 non-coding RNAs (ncRNAs) for a total of 20,802 transcripts. AmpliSeqTM yields highly precise and reproducible gene expression profiles (R2 >0.99). Taking advantage of AmpliSeq's reproducibility, we establish well-defined quantitative RNA expression patterns of HAM versus MDM, including significant M.tb-inducible genes, in networks and pathways that differ in part between MDM and HAM. A similar number of expressed genes are detected at all time-points between uninfected MDM and HAM, in common pathways including inflammatory and immune functions, but canonical pathway differences also exist. In particular, at 2 h, multiple genes relevant to the immune response are preferentially expressed in either uninfected HAM or MDM, while the HAM RNA profiles approximate MDM profiles over time in culture, highlighting the unique RNA expression profile of freshly obtained HAM. MDM demonstrate a greater transcriptional response than HAM upon M.tb infection, with 2 to >10 times more genes up- or down-regulated. The results identify key genes involved in cellular responses to M.tb in two different human macrophage types. Follow-up bioinformatics analysis indicates that approximately 30% of response genes have expression quantitative trait loci (eQTLs in GTEx), common DNA variants that can influence host gene expression susceptibility or resistance to M.tb, illustrated with the TREM1 gene cluster and IL-10.

Mycobacterium abscessus has emerged as an important pathogen in people with chronic inflammatory lung diseases such as cystic fibrosis, and recent reports suggest that it may be transmissible by fomites. M. abscessus exhibits two major colony morphology variants: a smooth morphotype (MaSm ) and a rough morphotype (MaRg ). Biofilm formation, prolonged intracellular survival, and colony variant diversity can each contribute to the persistence of M. abscessus and other bacterial pathogens in chronic pulmonary diseases. A prevailing paradigm of chronic M. abscessus infection is that MaSm is a noninvasive, biofilm-forming, persistent phenotype and MaRg an invasive phenotype that is unable to form biofilms. We show that MaRg is hyperaggregative and forms biofilm-like aggregates, which, like MaSm biofilm aggregates, are significantly more tolerant than planktonic variants to acidic pHs, hydrogen peroxide (H2O2), and treatment with amikacin or azithromycin. We further show that both variants are recalcitrant to antibiotic treatment inside human macrophage-like cells and that MaRg is more refractory than MaSm to azithromycin. Our results indicate that biofilm-like aggregation and protracted intracellular survival may each contribute to the persistence of this problematic pathogen in the face of antimicrobial agents regardless of morphotype. Biofilms of each M. abscessus variant are rapidly killed, however, by acetic acid, which may help to prevent local fomite transmission.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear transcription factor belonging to the superfamily of ligand-activated nuclear receptors. It is activated by diverse endogenous lipid metabolites as well as by exogenous ligands such as the thiazolidinediones. It regulates cellular metabolism, proliferation, differentiation, and inflammation, the latter in part through trans-repression of pro-inflammatory cytokines. PPARγ is highly expressed in alternatively activated alveolar macrophages (AMs), a primary host cell for airborne Mycobacterium tuberculosis (M.tb). Our previous in vitro study identified the importance of PPARγ activation through the mannose receptor (CD206) on human macrophages in enabling M. tb growth. The aim of the current study was to investigate the role of PPARγ in vivo during M. tb infection using a macrophage-specific PPARγ knock out mouse model with special emphasis on the lung environment. Our data show that the absence of PPARγ in lung macrophages reduces the growth of virulent M. tb, enhances pro-inflammatory cytokines and reduces granulomatous infiltration. These findings demonstrate that PPARγ activation, which down-regulates macrophage pro-inflammatory responses, impacts the lung's response to M. tb infection, thereby supporting PPARγ's role in tuberculosis (TB) pathogenesis.

Granulomas are the histopathologic hallmark of tuberculosis (TB), both in latency and active disease. Diagnostic and therapeutic strategies that specifically target granulomas have not been developed. Our objective is to develop a probe for imaging relevant immune cell populations infiltrating the granuloma. We report the binding specificity of Cyanine 3 (Cy3)-labeled cFLFLFK-PEG12 to human leukocytes and cellular constituents within a human in vitro granuloma model. We also report use of the probe in in vivo studies using a mouse model of lung granulomatous inflammation. We found that the probe preferentially binds human neutrophils and macrophages in human granuloma structures. Inhibition studies showed that peptide binding to human neutrophils is mediated by the receptor formyl peptide receptor 1 (FPR1). Imaging the distribution of intravenously administered cFLFLFK-PEG12-Cy3 in the mouse model revealed probe accumulation within granulomatous inflammatory responses in the lung. Further characterization revealed that the probe preferentially associated with neutrophils and cells of the monocyte/macrophage lineage. As there is no current clinical diagnostic imaging tool that specifically targets granulomas, the use of this probe in the context of latent and active TB may provide a unique advantage over current clinical imaging probes. We anticipate that utilizing a FPR1-targeted radiopharmaceutical analog of cFLFLFK in preclinical imaging studies may greatly contribute to our understanding of granuloma influx patterns and the biological roles and consequences of FPR1-expressing cells in contributing to disease pathogenesis.

The ability to grow at mammalian body temperatures is critical for pathogen infection of humans. For the thermally dimorphic fungal pathogen Histoplasma capsulatum, elevated temperature is required for differentiation of mycelia or conidia into yeast cells, a step critical for invasion and replication within phagocytic immune cells. Posttranslational glycosylation of extracellular proteins characterizes factors produced by the pathogenic yeast cells but not those of avirulent mycelia, correlating glycosylation with infection. Histoplasma yeast cells lacking the Pmt1 and Pmt2 protein mannosyltransferases, which catalyze O-linked mannosylation of proteins, are severely attenuated during infection of mammalian hosts. Cells lacking Pmt2 have altered surface characteristics that increase recognition of yeast cells by the macrophage mannose receptor and reduce recognition by the β-glucan receptor Dectin-1. Despite these changes, yeast cells lacking these factors still associate with and survive within phagocytes. Depletion of macrophages or neutrophils in vivo does not recover the virulence of the mutant yeast cells. We show that yeast cells lacking Pmt functions are more sensitive to thermal stress in vitro and consequently are unable to productively infect mice, even in the absence of fever. Treatment of mice with cyclophosphamide reduces the normal core body temperature of mice, and this decrease is sufficient to restore the infectivity of O-mannosylation-deficient yeast cells. These findings demonstrate that O-mannosylation of proteins increases the thermotolerance of Histoplasma yeast cells, which facilitates infection of mammalian hosts.IMPORTANCE For dimorphic fungal pathogens, mammalian body temperature can have contrasting roles. Mammalian body temperature induces differentiation of the fungal pathogen Histoplasma capsulatum into a pathogenic state characterized by infection of host phagocytes. On the other hand, elevated temperatures represent a significant barrier to infection by many microbes. By functionally characterizing cells lacking O-linked mannosylation enzymes, we show that protein mannosylation confers thermotolerance on H. capsulatum, enabling infection of mammalian hosts.

Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.

Despite its prominent role as a C-type lectin (CTL) pattern recognition receptor, mannose receptor (MR, CD206)-specific signaling molecules and pathways are unknown. The MR is highly expressed on human macrophages, regulating endocytosis, phagocytosis, and immune responses and mediating Mycobacterium tuberculosis (M.tb) phagocytosis by human macrophages, thereby limiting phagosome-lysosome (P-L) fusion. We identified human MR-associated proteins using phosphorylated and non-phosphorylated MR cytoplasmic tail peptides. We found that MR binds FcRγ-chain, which is required for MR plasma membrane localization and M.tb cell association. Additionally, we discovered that MR-mediated M.tb association triggers immediate MR tyrosine residue phosphorylation and Grb2 recruitment, activating the Rac/Pak/Cdc-42 signaling cascade important for M.tb uptake. MR activation subsequently recruits SHP-1 to the M.tb-containing phagosome, where its activity limits PI(3)P generation at the phagosome and M.tb P-L fusion and promotes M.tb growth. In sum, we identify human MR signaling pathways that temporally regulate phagocytosis and P-L fusion during M.tb infection.

Tuberculosis (TB) is a global epidemic caused by the infection of human macrophages with the world's most deadly single bacterial pathogen, Mycobacterium tuberculosis (M.tb). M.tb resides in a phagosomal niche within macrophages, where trace element concentrations impact the immune response, bacterial metal metabolism, and bacterial survival. The manipulation of micronutrients is a critical mechanism of host defense against infection. In particular, the human zinc transporter Zrt-/Irt-like protein 8 (ZIP8), one of 14 ZIP family members, is important in the flux of divalent cations, including zinc, into the cytoplasm of macrophages. It also has been observed to exist on the membrane of cellular organelles, where it can serve as an efflux pump that transports zinc into the cytosol. ZIP8 is highly inducible in response to M.tb infection of macrophages, and we have observed its localization to the M.tb phagosome. The expression, localization, and function of ZIP8 and other divalent cation transporters within macrophages have important implications for TB prevention and dissemination and warrant further study. In particular, given the importance of zinc as an essential nutrient required for humans and M.tb, it is not yet clear whether ZIP-guided zinc transport serves as a host protective factor or, rather, is targeted by M.tb to enable its phagosomal survival.

Macrophage intracellular pathogen killing is defective in cystic fibrosis (CF), despite abundant production of reactive oxygen species (ROS) in lung tissue. Burkholderia species can cause serious infection in CF and themselves affect key oxidase components in murine non-CF cells. However, it is unknown whether human CF macrophages have an independent defect in the oxidative burst and whether Burkholderia contributes to this defect in terms of assembly of the NADPH oxidase complex and subsequent ROS production. In this article, we analyze CF and non-CF human monocyte-derived macrophages (MDMs) for ROS production, NADPH assembly capacity, protein kinase C expression, and calcium release in response to PMA and CF pathogens. CF MDMs demonstrate a nearly 60% reduction in superoxide production after PMA stimulation compared with non-CF MDMs. Although CF MDMs generally have increased total NADPH component protein expression, they demonstrate decreased expression of the calcium-dependent protein kinase C conventional subclass α/β leading to reduced phosphorylation of NADPH oxidase components p47 phox and p40 phox in comparison with non-CF MDMs. Ingestion of B. cenocepacia independently contributes to and worsens the overall oxidative burst deficits in CF MDMs compared with non-CF MDMs. Together, these results provide evidence for inherent deficits in the CF macrophage oxidative burst caused by decreased phosphorylation of NADPH oxidase cytosolic components that are augmented by Burkholderia These findings implicate a critical role for defective macrophage oxidative responses in persistent bacterial infections in CF and create new opportunities for boosting the macrophage immune response to limit infection.

The mycobacterial cell wall is crucial to the host-pathogen interface, because it provides a barrier against antibiotics and the host immune response. In addition, cell wall lipids are mycobacterial virulence factors. The mycobacterial membrane protein large (MmpL) proteins are cell wall lipid transporters that are important for basic mycobacterial physiology and Mycobacterium tuberculosis pathogenesis. MmpL3 and MmpL11 are conserved across pathogenic and nonpathogenic mycobacteria, a feature consistent with an important role in the basic physiology of the bacterium. MmpL3 is essential and transports trehalose monomycolate to the mycobacterial surface. In this report, we characterize the role of MmpL11 in M. tuberculosis. M. tuberculosismmpL11 mutants have altered biofilms associated with lower levels of mycolic acid wax ester and long-chain triacylglycerols than those for wild-type bacteria. While the growth rate of the mmpL11 mutant is similar to that of wild-type M. tuberculosis in macrophages, the mutant exhibits impaired survival in an in vitro granuloma model. Finally, we show that the survival or recovery of the mmpL11 mutant is impaired when it is incubated under conditions of nutrient and oxygen starvation. Our results suggest that MmpL11 and its cell wall lipid substrates are important for survival in the context of adaptive immune pressure and for nonreplicating persistence, both of which are critically important aspects of M. tuberculosis pathogenicity.