Human Alveolar and Monocyte-Derived Human Macrophage Responses to Mycobacterium tuberculosis.

Alveolar macrophages (AMs) and recruited monocyte-derived macrophages (MDMs) mediate early lung immune responses to Mycobacterium tuberculosis. Differences in the response of these distinct cell types are poorly understood and may provide insight into mechanisms of tuberculosis pathogenesis. The objective of this study was to determine whether M. tuberculosis induces unique and essential antimicrobial pathways in human AMs compared with MDMs. Using paired human AMs and 5-d MCSF-derived MDMs from six healthy volunteers, we infected cells with M. tuberculosis H37Rv for 6 h, isolated RNA, and analyzed transcriptomic profiles with RNA sequencing. We found 681 genes that were M. tuberculosis dependent in AMs compared with MDMs and 4538 that were M. tuberculosis dependent in MDMs, but not AMs (false discovery rate [FDR] < 0.05). Using hypergeometric enrichment of DEGs in Broad Hallmark gene sets, we found that type I and II IFN Response were the only gene sets selectively induced in M. tuberculosis-infected AM (FDR < 0.05). In contrast, MYC targets, unfolded protein response and MTORC1 signaling, were selectively enriched in MDMs (FDR < 0.05). IFNA1, IFNA8, IFNE, and IFNL1 were specifically and highly upregulated in AMs compared with MDMs at baseline and/or after M. tuberculosis infection. IFNA8 modulated M. tuberculosis-induced proinflammatory cytokines and, compared with other IFNs, stimulated unique transcriptomes. Several DNA sensors and IFN regulatory factors had higher expression at baseline and/or after M. tuberculosis infection in AMs compared with MDMs. These findings demonstrate that M. tuberculosis infection induced unique transcriptional responses in human AMs compared with MDMs, including upregulation of the IFN response pathway and specific DNA sensors.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

Tn-Seq reveals hidden complexity in the utilization of host-derived glutathione in Francisella tularensis.

Host-derived glutathione (GSH) is an essential source of cysteine for the intracellular pathogen Francisella tularensis. In a comprehensive transposon insertion sequencing screen, we identified several F. tularensis genes that play central and previously unappreciated roles in the utilization of GSH during the growth of the bacterium in macrophages. We show that one of these, a gene we named dptA, encodes a proton-dependent oligopeptide transporter that enables growth of the organism on the dipeptide Cys-Gly, a key breakdown product of GSH generated by the enzyme γ-glutamyltranspeptidase (GGT). Although GGT was thought to be the principal enzyme involved in GSH breakdown in F. tularensis, our screen identified a second enzyme, referred to as ChaC, that is also involved in the utilization of exogenous GSH. However, unlike GGT and DptA, we show that the importance of ChaC in supporting intramacrophage growth extends beyond cysteine acquisition. Taken together, our findings provide a compendium of F. tularensis genes required for intracellular growth and identify new players in the metabolism of GSH that could be attractive targets for therapeutic intervention.

Nicotinamide Limits Replication of Mycobacterium tuberculosis and Bacille Calmette-Guérin Within Macrophages.

Novel antimicrobials for treatment of Mycobacterium tuberculosis are needed. We hypothesized that nicotinamide (NAM) and nicotinic acid (NA) modulate macrophage function to restrict M. tuberculosis replication in addition to their direct antimicrobial properties. Both compounds had modest activity in 7H9 broth, but only NAM inhibited replication in macrophages. Surprisingly, in macrophages NAM and the related compound pyrazinamide restricted growth of bacille Calmette-Guérin but not wild-type Mycobacterium bovis, which both lack a functional nicotinamidase/pyrazinamidase (PncA) rendering each strain resistant to these drugs in broth culture. Interestingly, NAM was not active in macrophages infected with a virulent M. tuberculosis mutant encoding a deletion in pncA. We conclude that the differential activity of NAM and nicotinic acid on infected macrophages suggests host-specific NAM targets rather than PncA-dependent direct antimicrobial properties. These activities are sufficient to restrict attenuated BCG, but not virulent wild-type M. bovis or M. tuberculosis.

Synthetic Macromolecular Antibiotic Platform for Inhalable Therapy against Aerosolized Intracellular Alveolar Infections.

Lung-based intracellular bacterial infections remain one of the most challenging infectious disease settings. For example, the current standard for treating Franciscella tularensis pneumonia (tularemia) relies on administration of oral or intravenous antibiotics that poorly achieve and sustain pulmonary drug bioavailability. Inhalable antibiotic formulations are approved and in clinical development for upper respiratory infections, but sustained drug dosing from inhaled antibiotics against alveolar intracellular infections remains a current unmet need. To provide an extended therapy against alveolar intracellular infections, we have developed a macromolecular therapeutic platform that provides sustained local delivery of ciprofloxacin with controlled dosing profiles. Synthesized using RAFT polymerization, these macromolecular prodrugs characteristically have high drug loading (16-17 wt % drug), tunable hydrolysis kinetics mediated by drug linkage chemistry (slow-releasing alkyllic vs fast-releasing phenolic esters), and, in general, represent new fully synthetic nanotherapeutics with streamlined manufacturing profiles. In aerosolized and completely lethal F.t. novicida mouse challenge models, the fast-releasing ciprofloxacin macromolecular prodrug provided high cure efficiencies (75% survival rate under therapeutic treatment), and the importance of release kinetics was demonstrated by the inactivity of the similar but slow-releasing prodrug system. Pharmacokinetics and biodistribution studies further demonstrated that the efficacious fast-releasing prodrug retained drug dosing in the lung above the MIC over a 48 h period with corresponding Cmax/MIC and AUC0-24h/MIC ratios being greater than 10 and 125, respectively; the thresholds for optimal bactericidal efficacy. These findings identify the macromolecular prodrug platform as a potential therapeutic system to better treat alveolar intracellular infections such as F. tularensis, where positive patient outcomes require tailored antibiotic pharmacokinetic and treatment profiles.

The role of NOD2 in murine and human melioidosis.

Nucleotide-binding oligomerization domain 2 (NOD2) is a cytosolic pathogen recognition receptor that regulates susceptibility to a variety of infections and chronic diseases. Burkholderia pseudomallei, a facultative intracellular bacterium, causes the tropical infection melioidosis. We hypothesized that NOD2 may participate in host defense in melioidosis. We performed a series of in vitro assays and in vivo experiments and analyzed the association of human genetic variation with infection to delineate the contribution of NOD2 to the host response to B. pseudomallei. We found that transfection with NOD2 mediated NF-κB activation induced by B. pseudomallei stimulation of HEK293 cells. After low-dose inoculation with aerosolized B. pseudomallei, Nod2-deficient mice showed impaired clinical responses and permitted greater bacterial replication in the lung and dissemination to the spleen compared with wild-type mice. IL-6 and KC levels were higher in the lungs of Nod2-deficient mice. In a cohort of 1562 Thai subjects, a common genetic polymorphism in the NOD2 region, rs7194886, was associated with melioidosis, and this effect was most pronounced in women. rs7194886 was not associated with differences in cytokine production induced by whole-blood stimulation with the NOD2 ligand, muramyl dipeptide, or B. pseudomallei. To our knowledge, these findings are the first to characterize the role of NOD2 in host defense in mammalian melioidosis.

Prior infection with Type A Francisella tularensis antagonizes the pulmonary transcriptional response to an aerosolized Toll-like receptor 4 agonist.

BACKGROUND: Francisella infection attenuates immune cell infiltration and expression of selected pro-inflammatory cytokines in response to endogenous LPS, suggesting the bacteria is actively antagonizing at least some part of the response to Toll-like receptor 4 (TLR4) engagement. The ability of different Francisella strains to inhibit the ability of E. coli LPS to induce a pulmonary inflammatory response, as measured by gene expression profiling, was examined to define the scope of modulation and identify of inflammatory genes/pathways that are specifically antagonized by a virulent F. tularensis infection. RESULTS: Prior aerosol exposure to F. tularensis subsp. tularensis, but not the live attenuated strain (LVS) of F. tularensis subsp. holarctica or F. novicida, significantly antagonized the transcriptional response in the lungs of infected mice exposed to aerosolized E. coli LPS. The response to E. coli LPS was not completely inhibited, suggesting that the bacteria is targeting further downstream of the TLR4 molecule. Analysis of the promotors of LPS-responsive genes that were perturbed by Type A Francisella infection identified candidate transcription factors that were potentially modulated by the bacteria, including multiple members of the forkhead transcription factor family (FoxA1, Foxa2, FoxD1, Foxd3, Foxf2, FoxI1, Fox03, Foxq1), IRF1, CEBPA, and Mef2. The annotated functional roles of the affected genes suggested that virulent Francisella infection suppressed cellular processes including mRNA processing, antiviral responses, intracellular trafficking, and regulation of the actin cytoskeleton. Surprisingly, despite the broad overall suppression of LPS-induced genes by virulent Francisella, and contrary to what was anticipated from prior studies, Type A Francisella did not inhibit the expression of the majority of LPS-induced cytokines, nor the expression of many classic annotated inflammatory genes. CONCLUSIONS: Collectively, this analysis demonstrates clear differences in the ability of different Francisella strains to modulate TLR4 signaling and identifies genes/pathways that are specifically targeted by virulent Type A Francisella.

Impaired TLR5 functionality is associated with survival in melioidosis.

Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR5(1174C)>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney-293 cells transfected with TLR5 and found that B. pseudomallei induced TLR5(1174C)- but not TLR5(1174T)-dependent activation of NF-κB. We tested the association of TLR5(1174C)>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR5(1174C)>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08-0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR5(1174C)>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1ß in carriers of TLR5(1174T) compared with carriers of TLR5(1174C). B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR5(1174T). We conclude that the hypofunctional genetic variant TLR5(1174C)>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis.

VgrG-5 is a Burkholderia type VI secretion system-exported protein required for multinucleated giant cell formation and virulence.

The type VI secretion system (T6SS) has emerged as a critical virulence factor for the group of closely related Burkholderia spp. that includes Burkholderia pseudomallei, B. mallei, and B. thailandensis. While the genomes of these bacteria, referred to as the Bptm group, appear to encode several T6SSs, we and others have shown that one of these, type VI secretion system 5 (T6SS-5), is required for virulence in mammalian infection models. Despite its pivotal role in the pathogenesis of the Bptm group, the effector repertoire of T6SS-5 has remained elusive. Here we used quantitative mass spectrometry to compare the secretome of wild-type B. thailandensis to that of a mutant harboring a nonfunctional T6SS-5. This analysis identified VgrG-5 as a novel secreted protein whose export depends on T6SS-5 function. Bioinformatics analysis revealed that VgrG-5 is a specialized VgrG protein that harbors a C-terminal domain (CTD) conserved among Bptm group species. We found that a vgrG-5 ΔCTD mutant is avirulent in mice and is unable to stimulate the fusion of host cells, a hallmark of the Bptm group previously shown to require T6SS-5 function. The singularity of VgrG-5 as a detected T6SS-5 substrate, taken together with the essentiality of its CTD for virulence, suggests that the protein is critical for the effector activity of T6SS-5. Intriguingly, we show that unlike the bacterial-cell-targeting T6SSs characterized so far, T6SS-5 localizes to the bacterial cell pole. We propose a model whereby the CTD of VgrG-5-, propelled by T6SS-5-, plays a key role in inducing membrane fusion, either by the recruitment of other factors or by direct participation.

Nucleotide-binding oligomerization domain containing-like receptor family, caspase recruitment domain (CARD) containing 4 (NLRC4) regulates intrapulmonary replication of aerosolized Legionella pneumophila.

BACKGROUND: Legionella pneumophila (Lp) flagellin activates signaling pathways in murine macrophages that control Lp replication. Nucleotide-binding oligomerization domain (NOD) containing-like receptor (NLR) family, caspase recruitment domain (CARD) containing 4 (NLRC4) and Toll-like Receptor (TLR5) both recognize Lp flagellin in vitro, but whether these two receptors play redundant or separate functional roles in vivo is unknown. METHODS: The immune response of Nlrc4-/-, Nlrc4-/-/Tlr5-/-, and wild type C57Bl/6 mice was analyzed after in vivo infection with aerosolized Lp. RESULTS: Lp clearance from the lungs was delayed in Nlrc4-/- mice over seven days in comparison to wild type controls. Nlrc4-/-/Tlr5-/- mice had no additional defect. In contrast to TLR5, NLRC4 did not regulate recruitment of neutrophils to the lung. Although there were no differences among the mouse strains in the lung transcriptome at 4 hours, Nlrc4-/- and Nlrc4-/-Tlr5-/- mice had increased lung inflammation at 72 hours in comparison to WT. Nlrc4-/-/Tlr5-/- mice also had altered cytokine production at both 4 and 24 hours post infection when compared to wild-type (WT) and Nlrc4-/- mice. Lp replication in murine alveolar macrophages was NLRC4-dependent and TLR5-independent. CONCLUSION: These studies reveal that NLRC4 and TLR5 mediate different roles in the inflammatory response to Lp flagellin in an aerosolized infection model and NLRC4 regulates replication in both lungs and alveolar macrophages.

Discovery of a glutathione utilization pathway in Francisella that shows functional divergence between environmental and pathogenic species.

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

Proteomic analysis of bronchoalveolar lavage fluid proteins from mice infected with Francisella tularensis ssp. novicida.

Francisella tularensis causes the zoonosis tularemia in humans and is one of the most virulent bacterial pathogens. We utilized a global proteomic approach to characterize protein changes in bronchoalveolar lavage fluid from mice exposed to one of three organisms, F. tularensis ssp. novicida, an avirulent mutant of F. tularensis ssp. novicida (F.t. novicida-ΔmglA), and Pseudomonas aeruginosa. The composition of bronchoalveolar lavage fluid (BALF) proteins was altered following infection, including proteins involved in neutrophil activation, oxidative stress, and inflammatory responses. Components of the innate immune response were induced including the acute phase response and the complement system; however, the timing of their induction varied. F. tularensis ssp. novicida infected mice do not appear to have an effective innate immune response in the first hours of infection; however, within 24 h, they show an upregulation of innate immune response proteins. This delayed response is in contrast to P. aeruginosa infected animals which show an early innate immune response. Likewise, F.t. novicida-ΔmglA infection initiates an early innate immune response; however, this response is diminished by 24 h. Finally, this study identifies several candidate biomarkers, including Chitinase 3-like-1 (CHI3L1 or YKL-40) and peroxiredoxin 1, that are associated with F. tularensis ssp. novicida but not P. aeruginosa infection.

Role of Francisella lipid A phosphate modification in virulence and long-term protective immune responses.

Lipopolysaccharide (LPS) structural modifications have been shown to specifically affect the pathogenesis of many gram-negative pathogens. In Francisella, modification of the lipid A component of LPS resulted in a molecule with no to low endotoxic activity. The role of the terminal lipid A phosphates in host recognition and pathogenesis was determined using a Francisella novicida mutant that lacked the 4' phosphatase enzyme (LpxF). The lipid A of this strain retained the phosphate moiety at the 4' position and the N-linked fatty acid at the 3' position on the diglucosamine backbone. Studies were undertaken to determine the pathogenesis of this mutant strain via the pulmonary and subcutaneous routes of infection. Mice infected with the lpxF-null F. novicida mutant by either route survived primary infection and subsequently developed protective immunity against a lethal wild-type (WT) F. novicida challenge. To determine the mechanism(s) by which the host controlled primary infection by the lpxF-null mutant, the role of innate immune components, including Toll-like receptor 2 (TLR2), TLR4, caspase-1, MyD88, alpha interferon (IFN-α), and gamma interferon(IFN-γ), was examined using knockout mice. Interestingly, only the IFN-γ knockout mice succumbed to a primary lpxF-null F. novicida mutant infection, highlighting the importance of IFN-γ production. To determine the role of components of the host adaptive immune system that elicit the long-term protective immune response, T- and B-cell deficient RAG1(-/-) mice were examined. All mice survived primary infection; however, RAG1(-/-) mice did not survive WT challenge, highlighting a role for T and B cells in the protective immune response.

Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.

Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.

Murine pulmonary infection and inflammation induced by inhalation of Burkholderia pseudomallei.

Melioidosis is a tropical disease caused by ingestion, percutaneous inoculation or inhalation of the Gram-negative soil saprophyte Burkholderia pseudomallei. We developed a reproducible experimental murine model of pneumonic melioidosis induced by inhalation of aerosolized B. pseudomallei 1026b. In a series of experiments performed to bracket the lethal dose, we found that C57BL/6 mice were modestly more resistant than BALB/c mice (median lethal dose 334 CFU/lung vs 204 CFU/lung). We further characterized infection and pulmonary inflammation in C57BL/6 mice infected with a sublethal dose. We observed pulmonary replication and dissemination of bacteria to distant organs in the first days after infection, followed by bacterial containment by day 4 and no evidence of recrudescent infection for up to 2 months. We measured a robust host inflammatory response notable for a neutrophilic bronchoalveolar lavage fluid profile, elevated cytokines and chemokines in the lung and serum and scattered foci of neutrophilic infiltrates in the alveoli and in a perivascular distribution on histological analysis. We previously noted a similar pattern of inflammation in mice infected with aerosolized B. thailandensis. This report builds on the limited literature describing experimental murine pneumonic melioidosis induced by aerosol and characterizes pulmonary infection and resultant inflammation in C57BL/6 mice infected with aerosolized B. pseudomallei. This model has utility for the study of bacterial and host factors that contribute to the virulence of melioidosis.

NOD1 and NOD2 regulation of pulmonary innate immunity to Legionella pneumophila.

The role of nucleotide-binding oligomerization domain-1 (NOD1) and nucleotide-binding oligomerization domain-2 (NOD2), cytoplasmic receptors which detect bacterial cell wall molecules, in pulmonary innate immune responses is poorly understood. We determined that both NOD1 and NOD2 detect heat-killed Legionella and stimulate NF-κb and IFN-ß promoter activity using an in vitro luciferase reporter system. We next infected NOD1- and NOD2-deficient animals with aerosolized Legionella pneumophila. At 3 days post infection, Nod1(-/-) mice had impaired bacterial clearance compared to WT controls. In addition, at 4 h and 24 h, Nod1(-/-) mice had impaired neutrophil recruitment to the alveolar space. In contrast, increased lung neutrophils were seen in the Nod2(-/-) animals at 24 h. Analysis of cytokine production at 4 h post infection revealed a significant decrease in proinflammatory cytokines in the Nod1(-/-) animals when compared to WT animals. In contrast, increased 4-h proinflammatory cytokines were seen in the Nod2(-/-) animals. Furthermore, the lungs of both Nod1(-/-) and Nod2(-/-) mice had significantly increased pro-inflammatory cytokine levels at 24 h, suggesting possible suppressive roles for later stages of infection. Together, our data suggest that although both NOD1 and NOD2 can detect Legionella, these receptors modulate the in vivo pulmonary immune response differently.

Epilysin (MMP-28) restrains early macrophage recruitment in Pseudomonas aeruginosa pneumonia.

Several members of the matrix metalloproteinase (MMP) family function in various processes of innate immunity, particularly in controlling leukocyte influx. Epilysin (MMP-28) is expressed in numerous tissues and, in adult mice, it has the highest expression in lung, where it is detected in bronchial epithelial cells (Clara cells). Epilysin is also expressed by bone marrow-derived macrophages, but not by alveolar macrophages, suggesting that its expression by macrophages is dependent on localization and differentiation. To assess the role of this MMP, we generated epilysin-null (Mmp28(-/-)) mice. Although epilysin is constitutively expressed in normal tissues, Mmp28(-/-) mice have no overt phenotype. However, using a murine model of Pseudomonas aeruginosa pneumonia, we found that Mmp28(-/-) mice had an early increase in macrophage recruitment into the lungs, as well as enhanced bacterial clearance and reduced pulmonary neutrophilia, which we predicted were due to accelerated macrophage influx. Macrophage depletion in WT and Mmp28(-/-) mice confirmed a role for macrophages in clearing P. aeruginosa and regulating neutrophil recruitment. Furthermore, we observed that macrophages derived from Mmp28(-/-) mice migrated faster than did wild-type cells to bronchoalveolar lavage fluid from P. aeruginosa-treated mice of either genotype. These observations indicate that epilysin functions as an intrinsic negative regulator of macrophage recruitment by retarding the chemotaxis of these cells.

Glycated Albumin Triggers an Inflammatory Response in the Human Airway Epithelium and Causes an Increase in Ciliary Beat Frequency.

The role of inflammation in airway epithelial cells and its regulation are important in several respiratory diseases. When disease is present, the barrier between the pulmonary circulation and the airway epithelium is damaged, allowing serum proteins to enter the airways. We identified that human glycated albumin (GA) is a molecule in human serum that triggers an inflammatory response in human airway epithelial cultures. We observed that single-donor human serum induced IL-8 secretion from primary human airway epithelial cells and from a cystic fibrosis airway cell line (CF1-16) in a dose-dependent manner. IL-8 secretion from airway epithelial cells was time dependent and rapidly increased in the first 4 h of incubation. Stimulation with GA promoted epithelial cells to secrete IL-8, and this increase was blocked by the anti-GA antibody. The IL-8 secretion induced by serum GA was 10-50-fold more potent than TNFα or LPS stimulation. GA also has a functional effect on airway epithelial cells in vitro, increasing ciliary beat frequency. Our results demonstrate that the serum molecule GA is pro-inflammatory and triggers host defense responses including increases in IL-8 secretion and ciliary beat frequency in the human airway epithelium. Although the binding site of GA has not yet been described, it is possible that GA could bind to the receptor for advanced glycated end products (RAGE), known to be expressed in the airway epithelium; however, further experiments are needed to identify the mechanism involved. We highlight a possible role for GA in airway inflammation.

HDAC3 inhibitor RGFP966 controls bacterial growth and modulates macrophage signaling during Mycobacterium tuberculosis infection.

RATIONALE: Host-directed therapeutics for Mycobacterium tuberculosis (Mtb) offer potential strategies for combatting antibiotic resistance and for killing non-replicating bacilli. Phenylbutyrate, a partially selective histone-deacetylase (HDAC) inhibitor, was previously shown to control Mtb growth and alter macrophage inflammatory pathways at 2-4 mM concentrations. OBJECTIVE: To identify a more potent and selective HDAC inhibitor that modulates macrophage responses to mycobacteria and has direct antibacterial effects against Mtb. METHODS: We used cellular approaches to characterize the role of pharmacologic inhibition of HDAC3 on Mtb growth and Mtb-induced peripheral and alveolar macrophage immune functions. MEASUREMENTS AND MAIN RESULTS: RGFP966, an HDAC3 inhibitor, controlled Mtb, BCG and M. avium growth directly in broth culture and in human peripheral blood monocyte-derived and alveolar macrophages with an MIC50 of approximately 5-10 µM. In contrast, RGFP966 did not inhibit growth of several other intracellular and extracellular bacteria. We also found that RGFP966 modulated macrophage pro-inflammatory cytokine secretion in response to Mtb infection with decreased IL6 and TNF secretion. CONCLUSIONS: We identified a potent and selective small molecule inhibitor of HDAC3 with direct antimicrobial activity against Mtb and modulation of macrophage signaling pathways.