Throat microbiota drives alterations in pulmonary alveolar microbiota in patients with septic ARDS.

OBJECTIVES: The translocation of intestinal flora has been linked to the colonization of diverse and heavy lower respiratory flora in patients with septic ARDS, and is considered a critical prognostic factor for patients. METHODS: On the first and third days of ICU admission, BALF, throat swab, and anal swab were collected, resulting in a total of 288 samples. These samples were analyzed using 16S rRNA analysis and the traceability analysis of new generation technology. RESULTS: On the first day, among the top five microbiota species in abundance, four species were found to be identical in BALF and throat samples. Similarly, on the third day, three microbiota species were found to be identical in abundance in both BALF and throat samples. On the first day, 85.16% of microorganisms originated from the throat, 5.79% from the intestines, and 9.05% were unknown. On the third day, 83.52% of microorganisms came from the throat, 4.67% from the intestines, and 11.81% were unknown. Additionally, when regrouping the 46 patients, the results revealed a significant predominance of throat microorganisms in BALF on both the first and third day. Furthermore, as the disease progressed, the proportion of intestinal flora in BALF increased in patients with enterogenic ARDS. CONCLUSIONS: In patients with septic ARDS, the main source of lung microbiota is primarily from the throat. Furthermore, the dynamic trend of the microbiota on the first and third day is essentially consistent.It is important to note that the origin of the intestinal flora does not exclude the possibility of its origin from the throat.

Surrogate infection model predicts optimal alveolar macrophage number for clearance of Aspergillus fumigatus infections.

The immune system has to fight off hundreds of microbial invaders every day, such as the human-pathogenic fungus Aspergillus fumigatus. The fungal conidia can reach the lower respiratory tract, swell and form hyphae within six hours causing life-threatening invasive aspergillosis. Invading pathogens are continuously recognized and eliminated by alveolar macrophages (AM). Their number plays an essential role, but remains controversial with measurements varying by a factor greater than ten for the human lung. We here investigate the impact of the AM number on the clearance of A. fumigatus conidia in humans and mice using analytical and numerical modeling approaches. A three-dimensional to-scale hybrid agent-based model (hABM) of the human and murine alveolus allowed us to simulate millions of virtual infection scenarios, and to gain quantitative insights into the infection dynamics for varying AM numbers and infection doses. Since hABM simulations are computationally expensive, we derived and trained an analytical surrogate infection model on the large dataset of numerical simulations. This enables reducing the number of hABM simulations while still providing (i) accurate and immediate predictions on infection progression, (ii) quantitative hypotheses on the infection dynamics under healthy and immunocompromised conditions, and (iii) optimal AM numbers for combating A. fumigatus infections in humans and mice.

Early alveolar macrophage response and IL-1R-dependent T cell priming determine transmissibility of Mycobacterium tuberculosis strains.

Mechanisms underlying variability in transmission of Mycobacterium tuberculosis strains remain undefined. By characterizing high and low transmission strains of M.tuberculosis in mice, we show here that high transmission M.tuberculosis strain induce rapid IL-1R-dependent alveolar macrophage migration from the alveolar space into the interstitium and that this action is key to subsequent temporal events of early dissemination of bacteria to the lymph nodes, Th1 priming, granulomatous response and bacterial control. In contrast, IL-1R-dependent alveolar macrophage migration and early dissemination of bacteria to lymph nodes is significantly impeded in infection with low transmission M.tuberculosis strain; these events promote the development of Th17 immunity, fostering neutrophilic inflammation and increased bacterial replication. Our results suggest that by inducing granulomas with the potential to develop into cavitary lesions that aids bacterial escape into the airways, high transmission M.tuberculosis strain is poised for greater transmissibility. These findings implicate bacterial heterogeneity as an important modifier of TB disease manifestations and transmission.

Targeting Intra-Pulmonary P53-Dependent Long Non-Coding RNA Expression as a Therapeutic Intervention for Systemic Lupus Erythematosus-Associated Diffuse Alveolar Hemorrhage.

Diffuse alveolar hemorrhage (DAH) in systemic lupus erythematosus (SLE) is associated with significant mortality, requiring a thorough understanding of its complex mechanisms to develop novel therapeutics for disease control. Activated p53-dependent apoptosis with dysregulated long non-coding RNA (lncRNA) expression is involved in the SLE pathogenesis and correlated with clinical activity. We examined the expression of apoptosis-related p53-dependent lncRNA, including H19, HOTAIR and lincRNA-p21 in SLE-associated DAH patients. Increased lincRNA-p21 levels were detected in circulating mononuclear cells, mainly in CD4+ and CD14+ cells. Higher expression of p53, lincRNA-p21 and cell apoptosis was identified in lung tissues. Lentivirus-based short hairpin RNA (shRNA)-transduced stable transfectants were created for examining the targeting efficacy in lncRNA. Under pristane stimulation, alveolar epithelial cells had increased p53, lincRNA-p21 and downstream Bax levels with elevated apoptotic ratios. After pristane injection, C57/BL6 mice developed DAH with increased pulmonary expression of p53, lincRNA-p21 and cell apoptosis. Intra-pulmonary delivery of shRNA targeting lincRNA-p21 reduced hemorrhage frequencies and improved anemia status through decreasing Bax expression and cell apoptosis. Our findings demonstrate increased p53-dependent lncRNA expression with accelerated cell apoptosis in the lungs of SLE-associated DAH patients, and show the therapeutic potential of targeting intra-pulmonary lncRNA expression in a pristane-induced model of DAH.

Comparative analysis of the bronchoalveolar microbiome in Portuguese patients with different chronic lung disorders.

The lung is inhabited by a diverse microbiome that originates from the oropharynx by a mechanism of micro-aspiration. Its bacterial biomass is usually low; however, this condition shifts in lung cancer (LC), chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD). These chronic lung disorders (CLD) may coexist in the same patient as comorbidities and share common risk factors, among which the microbiome is included. We characterized the microbiome of 106 bronchoalveolar lavages. Samples were initially subdivided into cancer and non-cancer and high-throughput sequenced for the 16S rRNA gene. Additionally, we used a cohort of 25 CLD patients where crossed comorbidities were excluded. Firmicutes, Proteobacteria and Bacteroidetes were the most prevalent phyla independently of the analyzed group. Streptococcus and Prevotella were associated with LC and Haemophilus was enhanced in COPD versus ILD. Although no significant discrepancies in microbial diversity were observed between cancer and non-cancer samples, statistical tests suggested a gradient across CLD where COPD and ILD displayed the highest and lowest alpha diversities, respectively. Moreover, COPD and ILD were separated in two clusters by the unweighted UniFrac distance (P value = 0.0068). Our results support the association of Streptoccocus and Prevotella with LC and of Haemophilus with COPD, and advocate for specific CLD signatures.

Proteomic Analysis of Humoral Immune Components in Bronchoalveolar Lavage of Patients Infected or Colonized by Aspergillus fumigatus.

Humoral immune components have been individually studied in the context of interaction of host with Aspergillus fumigatus, a major airborne fungal pathogen. However, a global view of the multitude and complex nature of humoral immune components is needed to bring new insight into host-Aspergillus interaction. Therefore, we undertook comparative proteomic analysis of the bronchoalveolar lavage fluid collected from individuals infected or colonized with A. fumigatus versus controls, to identify those alveolar humoral components affected upon A. fumigatus infection. Complement proteins C1q, C8 beta-chain, factor-H, ficolin-1, ficolin-2, mannan binding lectin serine peptidase 2, pentraxin-3 and the surfactant protein-D were identified as the major humoral immune components affected by A. fumigatus infection and colonization. Based on this observation, we hypothesize that crosstalk between these humoral components is essential during host-Aspergillus interaction giving new specific leads to study for better understanding the pathogenesis. Furthermore, the affected humoral components could be potential diagnostic markers of A. fumigatus infection or colonization.

Porphyromonas gingivalis enhances pneumococcal adhesion to human alveolar epithelial cells by increasing expression of host platelet-activating factor receptor.

Streptococcus pneumoniae causes pneumonia by infecting the alveolar epithelium via binding to host receptors, such as the platelet-activating factor receptor (PAFR). Although chronic periodontitis has been identified as a pneumonia risk factor, how periodontopathic bacteria cause pneumonia is not known. We found that S. pneumoniae adhered to PAFR expressed on A549 human alveolar epithelial cells stimulated by Porphyromonas gingivalis culture supernatant, and this was abrogated by a PAFR-specific inhibitor. Among the major virulence factors of P. gingivalis [lipopolysaccharide (LPS), fimbriae and gingipains (Rgps and Kgp)], PAFR expression and pneumococcal adhesion were executed in an Rgp-dependent manner. LPS and fimbriae did not induce PAFR expression. Hence, our findings suggest that P. gingivalis enhances pneumococcal adhesion to human alveoli by inducing PAFR expression and that gingipains are responsible for this.

Biological subphenotypes of acute respiratory distress syndrome may not reflect differences in alveolar inflammation.

Biological subphenotypes have been identified in acute respiratory distress syndrome (ARDS) based on two parsimonious models: the "uninflamed" and "reactive" subphenotype (cluster-model) and "hypo-inflammatory" and "hyper-inflammatory" (latent class analysis (LCA) model). The distinction between the subphenotypes is mainly driven by inflammatory and coagulation markers in plasma. However, systemic inflammation is not specific for ARDS and it is unknown whether these subphenotypes also reflect differences in the alveolar compartment. Alveolar inflammation and dysbiosis of the lung microbiome have shown to be important mediators in the development of lung injury. This study aimed to determine whether the "reactive" or "hyper-inflammatory" biological subphenotype also had higher concentrations of inflammatory mediators and enrichment of gut-associated bacteria in the lung. Levels of alveolar inflammatory mediators myeloperoxidase (MPO), surfactant protein D (SPD), interleukin (IL)-1b, IL-6, IL-10, IL-8, interferon gamma (IFN-Æ´), and tumor necrosis factor-alpha (TNFα) were determined in the mini-BAL fluid. Key features of the lung microbiome were measured: bacterial burden (16S rRNA gene copies/ml), community diversity (Shannon Diversity Index), and community composition. No statistically significant differences between the "uninflamed" and "reactive" ARDS subphenotypes were found in a selected set of alveolar inflammatory mediators and key features of the lung microbiome. LCA-derived subphenotypes and stratification based on cause of ARDS (direct vs. indirect) showed similar profiles, suggesting that current subphenotypes may not reflect the alveolar host response. It is important for future research to elucidate the pulmonary biology within each subphenotype properly, which is arguably a target for intervention.

Antibiotic Exposure Is Not Associated With Clearance of Bronchoalveolar Growth: Results From a Prospective Ventilator-Associated Pneumonia Study.

BACKGROUND: Ventilator-associated pneumonia is poorly understood in trauma. Ventilated trauma patients can develop bacterial burden without symptoms; the factors that influence this are unknown. METHODS: Injured adults ventilated for > 2 days were enrolled. Mini-bronchoalveolar lavage was performed for 14 days or until extubation. Semi-quantitative cultures were blinded from clinicians. All cultures with > 104 colony forming units (CFU) were assessed for antibiotic exposure (ABXE) and spectrum of coverage. mBAL CFU was assessed daily. RESULTS: 60 patients were ventilated for 9 days (median). There were 75 with > 104 CFU. 46 had > 104 CFU and no ABXE on the sample day. 74% had clearance or a decrease (CoD) in CFU without ABXE. 29 had > 104 CFU and ABXE on the sample day. 19 had ABXE with pathogen coverage. 84% had CoD in CFU. 10 had ABXE with no spectrum of coverage. 1/10 had increased CFU and the remaining 9/10 CoD in CFU. The three groups were not statistically different on chi-squared analysis. CONCLUSION: Clearance of pathogens on surveillance cultures was unaffected by ABXE.

The Role of Age, Neutrophil Infiltration and Antibiotics Timing in the Severity of Streptococcus pneumoniae Pneumonia. Insights from a Multi-Level Mathematical Model Approach.

Bacterial pneumonia is one of the most prevalent infectious diseases and has high mortality in sensitive patients (children, elderly and immunocompromised). Although an infection, the disease alters the alveolar epithelium homeostasis and hinders normal breathing, often with fatal consequences. A special case is hospitalized aged patients, which present a high risk of infection and death because of the community acquired version of the Streptococcus pneumoniae pneumonia. There is evidence that early antibiotics treatment decreases the inflammatory response during pneumonia. Here, we investigate mechanistically this strategy using a multi-level mathematical model, which describes the 24 first hours after infection of a single alveolus from the key signaling networks behind activation of the epithelium to the dynamics of the local immune response. With the model, we simulated pneumonia in aged and young patients subjected to different antibiotics timing. The results show that providing antibiotics to elderly patients 8 h in advance compared to young patients restores in aged individuals the effective response seen in young ones. This result suggests the use of early, probably prophylactic, antibiotics treatment in aged hospitalized people with high risk of pneumonia.

The ex vivo perfused human lung is resistant to injury by high-dose S. pneumoniae bacteremia.

Few patients with bacteremia from a nonpulmonary source develop acute respiratory distress syndrome (ARDS). However, the mechanisms that protect the lung from injury in bacteremia have not been identified. We simulated bacteremia by adding Streptococcus pneumoniae to the perfusate of the ex vivo perfused human lung model. In contrast to a pneumonia model in which bacteria were instilled into the distal air spaces of one lobe, injection of high doses of S. pneumoniae into the perfusate was not associated with alveolar epithelial injury as demonstrated by low protein permeability of the alveolar epithelium, intact alveolar fluid clearance, and the absence of alveolar edema. Unexpectedly, the ex vivo human lung rapidly cleared large quantities of S. pneumoniae even though the perfusate had very few intravascular phagocytes and lacked immunoglobulins or complement. The bacteria were cleared in part by the small number of neutrophils in the perfusate, alveolar macrophages in the airspaces, and probably by interstitial pathways. Together, these findings identify one mechanism by which the lung and the alveolar epithelium are protected from injury in bacteremia.

Co-infection with Staphylococcus aureus after primary influenza virus infection leads to damage of the endothelium in a human alveolus-on-a-chip model.

Pneumonia is one of the most common infectious diseases worldwide. The influenza virus can cause severe epidemics, which results in significant morbidity and mortality. Beyond the virulence of the virus itself, epidemiological data suggest that bacterial co-infections are the major cause of increased mortality. In this context, Staphylococcus aureus represents a frequent causative bacterial pathogen. Currently available models have several limitations in the analysis of the pathogenesis of infections, e.g. some bacterial toxins strongly act in a species-specific manner. Human 2D mono-cell culture models often fail to maintain the differentiation of alveolus-specific functions. A detailed investigation of the underlying pathogenesis mechanisms requires a physiological interaction of alveolus-specific cell types. The aim of the present work was to establish a human in vitro alveolus model system composed of vascular and epithelial cell structures with cocultured macrophages resembling the human alveolus architecture and functions. We demonstrate that high barrier integrity maintained for up to 14 d in our model containing functional tissue-resident macrophages. We show that flow conditions and the presence of macrophages increased the barrier function. The infection of epithelial cells induced a high inflammatory response that spread to the endothelium. Although the integrity of the epithelium was not compromised by a single infection or co-infection, we demonstrated significant endothelial cell damage associated with loss of barrier function. We established a novel immune-responsive model that reflects the complex crosstalk between pathogens and host. The in vitro model allows for the monitoring of spatiotemporal spreading of the pathogens and the characterization of morphological and functional alterations attributed to infection. The alveolus-on-a-chip represents a promising platform for mechanistic studies of host-pathogen interactions and the identification of molecular and cellular targets of novel treatment strategies in pneumonia.

Gene expression changes in human bronchial epithelial cells (BEAS-2B) and human pulmonary alveolar epithelial cells (HPAEpiC) after interaction with Cladosporium sphaerospermum.

Cladosporium is one of the most abundant spore. Fungi of this genus can cause respiratory allergy and intrabronchial lesion. We studied the differential expression of host genes after the interaction of Cladosporium sphaerospermum conidia with Human Bronchial Epithelial Cells (BEAS-2B) and Human Pulmonary Alveolar Epithelial Cells (HPAEpiC). C. sphaerospermum conidia were harvested and co-cultured with BEAS-2B cells or HPAEpiC cells for 48 hours respectively. This culture duration was chosen as it was associated with high germination rate. RNA was extracted from two biological replicates per treatment. RNA of BEAS-2B cells was used to assess changes in gene expression using AffymetrixGeneChip® Human Transcriptome Array 2.0. After co-culture with Cladosporium spores, 68 individual genes were found differentially expressed (P ≤ 0.05) and up-regulated ≥ 1.5 folds while 75 genes were found differentially expressed at ≤ -1.5 folds compared with controls. Reverse transcription and qPCR were performed on the RNA collected from both BEAS-2B cells and HPAEpiC cells to validate the microarray results with 7 genes. Based on the findings, infected pulmonary epithelial cells exhibited an increase in cell death-related genes and genes associated with innate immunity.

Mycobacterium abscessus, an Emerging and Worrisome Pathogen among Cystic Fibrosis Patients.

Nontuberculous mycobacteria (NTM) have recently emerged as important pathogens among cystic fibrosis (CF) patients worldwide. Mycobacterium abscessus is becoming the most worrisome NTM in this cohort of patients and recent findings clarified why this pathogen is so prone to this disease. M. abscessus drug therapy takes up to 2 years and its failure causes an accelerated lung function decline. The M. abscessus colonization of lung alveoli begins with smooth strains producing glycopeptidolipids and biofilm, whilst in the invasive infection, "rough" mutants are responsible for the production of trehalose dimycolate, and consequently, cording formation. Human-to-human M. abscessus transmission was demonstrated among geographically separated CF patients by whole-genome sequencing of clinical isolates worldwide. Using a M. abscessus infected CF zebrafish model, it was demonstrated that CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction seems to have a specific role in the immune control of M. abscessus infections only. This pathogen is also intrinsically resistant to many drugs, thanks to its physiology and to the acquisition of new mechanisms of drug resistance. Few new compounds or drug formulations active against M. abscessus are present in preclinical and clinical development, but recently alternative strategies have been investigated, such as phage therapy and the use of ß-lactamase inhibitors.

Antifungal synergy of a topical triazole, PC945, with a systemic triazole against respiratory Aspergillus fumigatus infection.

Invasive pulmonary Aspergillosis is a leading cause of morbidity and mortality in immunosuppressed patients and treatment outcomes using oral antifungal triazoles remain suboptimal. Here we show that combining topical treatment using PC945, a novel inhaled triazole, with systemic treatment using known triazoles demonstrated synergistic antifungal effects against Aspergillus fumigatus (AF) in an in vitro human alveolus bilayer model and in the lungs of neutropenic immunocompromised mice. Combination treatment with apical PC945 and either basolateral posaconazole or voriconazole resulted in a synergistic interaction with potency improved over either compound as a monotherapy against both azole-susceptible and resistant AF invasion in vitro. Surprisingly there was little, or no synergistic interaction observed when apical and basolateral posaconazole or voriconazole were combined. In addition, repeated prophylactic treatment with PC945, but not posaconazole or voriconazole, showed superior effects to single prophylactic dose, suggesting tissue retention and/or accumulation of PC945. Furthermore, in mice infected with AF intranasally, 83% of animals treated with a combination of intranasal PC945 and oral posaconazole survived until day 7, while little protective effects were observed by either compound alone. Thus, the combination of a highly optimised topical triazole with oral triazoles potentially induces synergistic effects against AF infection.

Development of an aerosolized Mannheimia haemolytica experimental pneumonia model in clean-catch colostrum-deprived calves.

Mannheimia haemolytica is an important cause of bovine respiratory disease (BRD). BRD is usually a multifactorial disease with host factors and viral infections influencing pathogenesis. Previous studies that have attempted to experimentally induce pneumonia using aerosolized M. haemolytica alone have produced inconsistent results, yet an aerosol model would be useful to study the details of early infection and to investigate the role of innate defences in pathogenesis. The objective of these studies was to develop and characterize an aerosolized M. haemolytica disease model. In an initial study, conventionally raised calves with higher levels of antibody against M. haemolytica leukotoxin developed acute respiratory distress and diffuse alveolar damage, but did not develop bronchopneumonia, following challenge with M. haemolytica serotype 1. Clean-catch colostrum-deprived calves challenged with 1 × 1010 colony forming units of M. haemolytica serotype 1 consistently developed bronchopneumonia, with elevations in rectal temperature, serum haptoglobin, plasma fibrinogen, and blood neutrophils. Mannheimia haemolytica serotype 1 was consistently isolated from the nasal cavities and lungs of challenged calves. Despite distribution of aerosol and isolation of M. haemolytica in all lung lobes, gross lesions were mainly observed in the cranioventral area of lung. Gross and histologic lesions included neutrophilic bronchopneumonia and fibrinous pleuritis, with oat cells (necrotic neutrophils with streaming nuclei), and areas of coagulative necrosis, which are similar to lesions in naturally occurring BRD. Thus, challenge with M. haemolytica serotype 1 and use of clean-catch colostrum-deprived calves with low or absent antibody titres allowed development of an effective aerosol challenge model that induced typical clinical disease and lesions.

Lung Microbiota Contribute to Pulmonary Inflammation and Disease Progression in Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) causes considerable global morbidity and mortality, and its mechanisms of disease progression are poorly understood. Recent observational studies have reported associations between lung dysbiosis, mortality, and altered host defense gene expression, supporting a role for lung microbiota in IPF. However, the causal significance of altered lung microbiota in disease progression is undetermined. Objectives: To examine the effect of microbiota on local alveolar inflammation and disease progression using both animal models and human subjects with IPF. Methods: For human studies, we characterized lung microbiota in BAL fluid from 68 patients with IPF. For animal modeling, we used a murine model of pulmonary fibrosis in conventional and germ-free mice. Lung bacteria were characterized using 16S rRNA gene sequencing with novel techniques optimized for low-biomass sample load. Microbiota were correlated with alveolar inflammation, measures of pulmonary fibrosis, and disease progression. Measurements and Main Results: Disruption of the lung microbiome predicts disease progression, correlates with local host inflammation, and participates in disease progression. In patients with IPF, lung bacterial burden predicts fibrosis progression, and microbiota diversity and composition correlate with increased alveolar profibrotic cytokines. In murine models of fibrosis, lung dysbiosis precedes peak lung injury and is persistent. In germ-free animals, the absence of a microbiome protects against mortality. Conclusions: Our results demonstrate that lung microbiota contribute to the progression of IPF. We provide biological plausibility for the hypothesis that lung dysbiosis promotes alveolar inflammation and aberrant repair. Manipulation of lung microbiota may represent a novel target for the treatment of IPF.

Comparative Assessment of Aspergillosis by Virtual Infection Modeling in Murine and Human Lung.

Aspergillus fumigatus is a ubiquitous opportunistic fungal pathogen that can cause severe infections in immunocompromised patients. Conidia that reach the lower respiratory tract are confronted with alveolar macrophages, which are the resident phagocytic cells, constituting the first line of defense. If not efficiently removed in time, A. fumigatus conidia can germinate causing severe infections associated with high mortality rates. Mice are the most extensively used model organism in research on A. fumigatus infections. However, in addition to structural differences in the lung physiology of mice and the human host, applied infection doses in animal experiments are typically orders of magnitude larger compared to the daily inhalation doses of humans. The influence of these factors, which must be taken into account in a quantitative comparison and knowledge transfer from mice to humans, is difficult to measure since in vivo live cell imaging of the infection dynamics under physiological conditions is currently not possible. In the present study, we compare A. fumigatus infection in mice and humans by virtual infection modeling using a hybrid agent-based model that accounts for the respective lung physiology and the impact of a wide range of infection doses on the spatial infection dynamics. Our computer simulations enable comparative quantification of A. fumigatus infection clearance in the two hosts to elucidate (i) the complex interplay between alveolar morphometry and the fungal burden and (ii) the dynamics of infection clearance, which for realistic fungal burdens is found to be more efficiently realized in mice compared to humans.

Therapeutic effects of human mesenchymal stem cell microvesicles in an ex vivo perfused human lung injured with severe E. coli pneumonia.

BACKGROUND: We previously reported that microvesicles (MVs) released by human mesenchymal stem cells (MSC) were as effective as the cells themselves in both Escherichia coli lipopolysaccharide and live bacteria-induced acute lung injury (ALI) mice models. However, it remained unclear whether the biological effect of MSC MV can be applied to human ALI. METHODS: In the current study, we tested the therapeutic effects of MSC MVs in a well-established ex vivo perfused human model of bacterial pneumonia. Using human donor lungs not used for transplantation, we instilled E. coli bacteria intrabronchially and, 1 hour later, administered MSC MVs into the perfusate as therapy. RESULTS: After 6 hours, instillation of E. coli bacteria caused influx of inflammatory cells, which resulted in significant inflammation, lung protein permeability and pulmonary oedema formation. Administration of MSC MV significantly increased alveolar fluid clearance and reduced protein permeability and numerically lowered the bacterial load in the injured alveolus. The beneficial effect on bacterial killing was more pronounced with pretreatment of MSCs with a Toll-like receptor 3 agonist, polyinosinic:polycytidylic acid (Poly (I:C)), prior to the isolation of MVs. Isolated human alveolar macrophages had increased antimicrobial activity with MSC MV treatment in vitro as well. Although oxygenation and lung compliance levels were similar between injury and treatment groups, administration of MSC MVs numerically decreased median pulmonary artery pressure at 6 hours. CONCLUSIONS: In summary, MSC MVs increased alveolar fluid clearance and reduced lung protein permeability, and pretreatment with Poly (I:C) enhanced the antimicrobial activity of MVs in an ex vivo perfused human lung with severe bacteria pneumonia.

Inhalable poly(lactic-co-glycolic acid) (PLGA) microparticles encapsulating all-trans-Retinoic acid (ATRA) as a host-directed, adjunctive treatment for Mycobacterium tuberculosis infection.

Ending the tuberculosis (TB) epidemic by 2030 was recently listed in the United Nations (UN) Sustainable Development Goals alongside HIV/AIDS and malaria as it continues to be a major cause of death worldwide. With a significant proportion of TB cases caused by resistant strains of Mycobacterium tuberculosis (Mtb), there is an urgent need to develop new and innovative approaches to treatment. Since 1989, researchers have been assessing the anti-bacterial effects of the active metabolite of vitamin A, all trans-Retinoic acid (ATRA) solution, in Mtb models. More recently the antibacterial effect of ATRA has been shown to regulate the immune response to infection via critical gene expression, monocyte activation and the induction of autophagy leading to its application as a host-directed therapy (HDT). Inhalation is an attractive route for targeted treatment of TB, and therefore we have developed ATRA-loaded microparticles (ATRA-MP) within the inhalable size range (2.07 ±â€¯0.5 µm) offering targeted delivery of the encapsulated cargo (70.5 ±â€¯2.3%) to the site of action within the alveolar macrophage, which was confirmed by confocal microscopy. Efficient cellular delivery of ATRA was followed by a reduction in Mtb growth (H37Ra) in THP-1 derived macrophages evaluated by both the BACT/ALERT® system and enumeration of colony forming units (CFU). The antibacterial effect of ATRA-MP treatment was further assessed in BALB/c mice infected with the virulent strain of Mtb (H37Rv). ATRA-MP treatments significantly decreased the bacterial burden in the lungs alongside a reduction in pulmonary pathology following just three doses administered intratracheally. The immunomodulatory effects of targeted ATRA treatment in the lungs indicate a distinct yet effective mechanism of action amongst the formulations. This is the first study to-date of a controlled release ATRA treatment for TB suitable for inhalation that offers improved targeting of a HDT, retains antibacterial efficacy and improves pulmonary pathology compared to ATRA solution.