Stenotrophomonas maltophilia contributes to smoking-related emphysema through IRF1-triggered PANoptosis of alveolar epithelial cells.

Cigarette smoke (CS), the main source of indoor air pollution and the primary risk factor for respiratory diseases, contains chemicals that can perturb microbiota through antibiotic effects. Although smoking induces a disturbance of microbiota in the lower respiratory tract, whether and how it contributes to initiation or promotion of emphysema are not well clarified. Here, we demonstrated an aberrant microbiome in lung tissue of patients with smoking-related COPD. We found that Stenotrophomonas maltophilia (S. maltophilia) was expanded in lung tissue of patients with smoking-related COPD. We revealed that S. maltophilia drives PANoptosis in alveolar epithelial cells and represses formation of alveolar organoids through IRF1 (interferon regulatory factor 1). Mechanistically, IRF1 accelerated transcription of ZBP1 (Z-DNA Binding Protein 1) in S. maltophilia-infected alveolar epithelial cells. Elevated ZBP1 served as a component of the PANoptosome, which triggered PANoptosis in these cells. By using of alveolar organoids infected by S. maltophilia, we found that targeting of IRF1 mitigated S. maltophilia-induced injury of these organoids. Moreover, the expansion of S. maltophilia and the expression of IRF1 negatively correlated with the progression of emphysema. Thus, the present study provides insights into the mechanism of lung dysbiosis in smoking-related COPD, and presents a potential target for mitigation of COPD progression.

Effect of Hcp Iron Ion Regulation on the Interaction Between Acinetobacter baumannii With Human Pulmonary Alveolar Epithelial Cells and Biofilm Formation.

Acinetobacter baumannii is a type of bacterial nosocomial infection with severe drug resistance. Hemolysin co-regulated protein (Hcp) is a marker of activated type VI secretion system (T6SS), a key secretory system that promotes Gram-negative bacteria colonization, adhesion, and invasion of host cells. Hcp is also regulated by iron ions (Fe). In this study, an ATCC17978 hcp deletion strain (ATCC17978Δhcp), an hcp complement strain (ATCC17978Δhcp+ ), and an A. baumannii-green fluorescent protein (GFP) strain were constructed and used to investigate the role of hcp in bacterial adhesion to cells (human pulmonary alveolar epithelial cells (HPAEpiC)) and biofilm formation. Our results indicate that the inhibitory concentrations of the three A. baumannii strains (ATCC17978 wild type, ATCC17978Δhcp, and ATCC17978Δhcp+) were drug-sensitive strains. A. baumannii hcp gene and iron ions might be involved in promoting the formation of a biofilm and host-bacteria interaction. Iron ions affected the ability of A. baumannii to adhere to cells, as there was no significant difference in the bacterial numbers when assessing the adhesion of the three strains to HPAEpiC in the presence of iron ion concentrations of 0 µM (F = 3.1800, p = 0.1144), 25 µM (F = 2.067, p = 0.2075), 100 µM (F = 30.52, p = 0.0007), and 400 µM (F = 17.57, p = 0.0031). The three strains showed significant differences in their ability to adhere to HPAEpiC. The numbers of bacteria adhesion to HPAEpiC were ATCC17978Δhcp>ATCC17978Δhcp+>ATCC17978 in descending order. Hcp gene was positively regulated by iron ions in the bacteria-cells' co-culture. It is speculated that the effect of iron ions on the interaction between A. baumannii and HPAEpiC might be related to the transport function of hcp and bacterial immune escape mechanisms.

Exosomal miR-181a-5p reduce Mycoplasma gallisepticum (HS strain) infection in chicken by targeting PPM1B and activating the TLR2-mediated MyD88/NF-κB signaling pathway.

Mycoplasma gallisepticum (MG) is one of the most important pathogens that causes chronic respiratory disease (CRD) in chickens. Exosomes secreted from cells have been well demonstrated to deliver miRNAs to recipient cells to modulate cellular functions. The purpose of this study is to explore the underlying functions and mechanisms of exosomal miR-181a-5p in MG-HS infection. In this study, we found that miR-181a-5p expression in vivo and in vitro was significantly up-regulated after MG-HS infection. It was also upregulated in exosomes, which were derived from MG-HS-infected type-II pneumocytes cells (CP-II). In addition, exosomes secreted by MG-HS-infected CP-II were able to transfer miR-181a-5p to recipient chicken embryo fibroblast cells (DF-1), resulting in a significant upregulation of miR-181a-5p expression in recipient DF-1 cells. We further identified that Mg2+/Mn2+-dependent protein phosphatase 1B (PPM1B) was the target gene of miR-181a-5p. Overexpression of miR-181a-5p or knockdown of PPM1B activated the nuclear factor-κB (NF-κB) signaling pathway, whereas inhibition of miR-181a-5p and overexpression of PPM1B led to the opposite results. Besides, up-regulation of miR-181a-5p significantly increased the expression of toll-like receptor 2 (TLR2), myeloid differentiation factor 88 (MyD88), tumor necrosis factors alpha (TNF-α) and interleukin-1ß (IL-1ß), whereas inhibition of miR-181a-5p showed a contrary result. Up-regulation of miR-181a-5p promoted cell proliferation, cell cycle progression and inhibited apoptosis to resist MG-HS infection. Moreover, overexpression of miR-181a-5p significantly negative regulated the expression of Mycoplasma gallisepticum adhesin protein (pMGA1.2) by directly inhibiting PPM1B. Thus, we concluded that exosomal miR-181a-5p from CP-II cells activated the TLR2-mediated MyD88/NF-κB signaling pathways by directly targeting PPM1B to promote the expression of pro-inflammatory cytokines for defending against MG-HS infection in recipient DF-1 cells.

IL-17 stimulates neutrophils to release S100A8/A9 to promote lung epithelial cell apoptosis in Mycoplasma pneumoniae-induced pneumonia in children.

Mycoplasma pneumoniae-induced pneumonia (MPP) is a common cause of community-acquired respiratory tract infections, increasing risk of morbidity and mortality, in children. However, diagnosing early-stage MPP is difficult owing to the lack of good diagnostic methods. Here, we examined the protein profile of bronchoalveolar lavage fluid (BALF) and found that S100A8/A9 was highly expressed. Enzyme-linked immunosorbent assays used to assess protein levels in serum samples indicated that S100A8/A9 concentrations were also increased in serum obtained from children with MPP, with no change in S100A8/A9 levels in children with viral or bacterial pneumonia. In vitro, S100A8/A9 treatment significantly increased apoptosis in a human alveolar basal epithelial cell line (A549 cells). Bioinformatics analyses indicated that up-regulated S100A8/A9 proteins participated in the interleukin (IL)-17 signaling pathway. The origin of the increased S100A8/A9 was investigated in A549 cells and in neutrophils obtained from children with MPP. Treatment of neutrophils, but not of A549 cells, with IL-17A released S100A8/A9 into the culture medium. In summary, we demonstrated that S100A8/A9, possibly released from neutrophils, is a new potential biomarker for the clinical diagnosis of children MPP and involved in the development of this disease through enhancing apoptosis of alveolar basal epithelial cells.

Pertactin contributes to shedding and transmission of Bordetella bronchiseptica.

Whooping cough is resurging in the United States despite high vaccine coverage. The rapid rise of Bordetella pertussis isolates lacking pertactin (PRN), a key vaccine antigen, has led to concerns about vaccine-driven evolution. Previous studies showed that pertactin can mediate binding to mammalian cells in vitro and act as an immunomodulatory factor in resisting neutrophil-mediated clearance. To further investigate the role of PRN in vivo, we examined the functions of pertactin in the context of a more naturally low dose inoculation experimental system using C3H/HeJ mice that is more sensitive to effects on colonization, growth and spread within the respiratory tract, as well as an experimental approach to measure shedding and transmission between hosts. A B. bronchiseptica pertactin deletion mutant was found to behave similarly to its wild-type (WT) parental strain in colonization of the nasal cavity, trachea, and lungs of mice. However, the pertactin-deficient strain was shed from the nares of mice in much lower numbers, resulting in a significantly lower rate of transmission between hosts. Histological examination of respiratory epithelia revealed that pertactin-deficient bacteria induced substantially less inflammation and mucus accumulation than the WT strain and in vitro assays verified the effect of PRN on the induction of TNF-α by murine macrophages. Interestingly, only WT B. bronchiseptica could be recovered from the spleen of infected mice and were further observed to be intracellular among isolated splenocytes, indicating that pertactin contributes to systemic dissemination involving intracellular survival. These results suggest that pertactin can mediate interactions with immune cells and augments inflammation that contributes to bacterial shedding and transmission between hosts. Understanding the relative contributions of various factors to inflammation, mucus production, shedding and transmission will guide novel strategies to interfere with the reemergence of pertussis.

Complement receptor 3 mediates Aspergillus fumigatus internalization into alveolar epithelial cells with the increase of intracellular phosphatidic acid by activating FAK.

Complement receptor 3 (CD11b/CD18) is an important receptor that mediates adhesion, phagocytosis and chemotaxis in various immunocytes. The conidia of the medically-important pathogenic fungus, Aspergillus fumigatus can be internalized into alveolar epithelial cells to disseminate its infection in immunocompromised host; however, the role of CR3 in this process is poorly understood. In the present study, we investigated the potential role of CR3 on A. fumigatus internalization into type II alveolar epithelial cells and its effect on host intracellular PA content induced by A. fumigatus. We found that CR3 is expressed in alveolar epithelial cells and that human serum and bronchoalveolar lavage fluid (BALF) could improve A. fumigatus conidial internalization into A549 type II alveolar epithelial cell line and mouse primary alveolar epithelial cells, which were significantly inhibited by the complement C3 quencher and CD11b-blocking antibody. Serum-opsonization of swollen conidia, but not resting conidia led to the increase of cellular phosphatidic acid (PA) in A549 cells during infection. Moreover, both conidial internalization and induced PA production were interfered by CD11b-blocking antibody and dependent on FAK activity, but not Syk in alveolar epithelial cells. Overall, our results revealed that CR3 is a critical modulator of Aspergillus fumigatus internalization into alveolar epithelial cells.

Evaluation of Stable LifeAct-mRuby2- and LAMP1-NeonGreen Expressing A549 Cell Lines for Investigation of Aspergillus fumigatus Interaction with Pulmonary Cells.

Inhaled Aspergillus fumigatus spores can be internalized by alveolar type II cells. Cell lines stably expressing fluorescently labeled components of endocytic pathway enable investigations of intracellular organization during conidia internalization and measurement of the process kinetics. The goal of this report was to evaluate the methodological appliance of cell lines for studying fungal conidia internalization. We have generated A549 cell lines stably expressing fluorescently labeled actin (LifeAct-mRuby2) and late endosomal protein (LAMP1-NeonGreen) following an evaluation of cell-pathogen interactions in live and fixed cells. Our data show that the LAMP1-NeonGreen cell line can be used to visualize conidia co-localization with LAMP1 in live and fixed cells. However, caution is necessary when using LifeAct-mRuby2-cell lines as it may affect the conidia internalization dynamics.

The genetic feature and virulence determinant of highly virulent community-associated MRSA ST338-SCCmec Vb in China.

ST59 is the predominant pathotype of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in China. As a variant of ST59, there is relatively little known about the detailed information of ST338. To address this issue, here, we described thirteen ST338 CA-MRSA strains isolated from severe bloodstream infection cases, and focused on their epidemiology, genetic features and virulence potential. Phylogenetic analysis showed the earliest isolated strain of this study is likely a predecessor of recent ST338 lineage (after year of 2014). Furthermore, the phylogenetic reconstruction and time estimation suggested that ST338 evolved from ST59 in 1991. Notably, the carrying patten of virulence factors of all ST338 strains were similar, and the genomic islands νSaα, νSaγ and SaPI and the core virulence factors like hla and psm were detected in ST338 isolates. However, all ST338 isolates lacked some adhesion factors such as clfA, clfB, eap, cna and icaD. Additionally, among these ST338 strains, one PVL-negative ST338 isolate was detected. Experiment on mice nose and human alveolar epithelial cell showed that the nasal colonization ability of ST338 was weaker than that of CA-MRSA MW2. In a mouse bloodstream infection model and skin infection model, PVL+ and PVL- strains had the similar virulence, which was dependent on upregulation of toxin genes rather than the presence of mobile genetic elements such as ΦSa2 carrying PVL. Our findings provide important insight into the epidemiology and pathogenicity of the novel and highly virulent ST338-SCCmec Vb clone.

Bronchial epithelial DNA methyltransferase 3b dampens pulmonary immune responses during Pseudomonas aeruginosa infection.

DNA methyltransferase (Dnmt)3b mediates de novo DNA methylation and modulation of Dnmt3b in respiratory epithelial cells has been shown to affect the expression of multiple genes. Respiratory epithelial cells provide a first line of defense against pulmonary pathogens and play a crucial role in the immune response during pneumonia caused by Pseudomonas (P.) aeruginosa, a gram-negative bacterium that expresses flagellin as an important virulence factor. We here sought to determine the role of Dntm3b in respiratory epithelial cells in immune responses elicited by P. aeruginosa. DNMT3B expression was reduced in human bronchial epithelial (BEAS-2B) cells as well as in primary human and mouse bronchial epithelial cells grown in air liquid interface upon exposure to P. aeruginosa (PAK). Dnmt3b deficient human bronchial epithelial (BEAS-2B) cells produced more CXCL1, CXCL8 and CCL20 than control cells when stimulated with PAK, flagellin-deficient PAK (PAKflic) or flagellin. Dnmt3b deficiency reduced DNA methylation at exon 1 of CXCL1 and enhanced NF-ĸB p65 binding to the CXCL1 promoter. Mice with bronchial epithelial Dntm3b deficiency showed increased Cxcl1 mRNA expression in bronchial epithelium and CXCL1 protein release in the airways during pneumonia caused by PAK, which was associated with enhanced neutrophil recruitment and accelerated bacterial clearance; bronchial epithelial Dnmt3b deficiency did not modify responses during pneumonia caused by PAKflic or Klebsiella pneumoniae (an un-flagellated gram-negative bacterium). Dnmt3b deficiency in type II alveolar epithelial cells did not affect mouse pulmonary defense against PAK infection. These results suggest that bronchial epithelial Dnmt3b impairs host defense during Pseudomonas induced pneumonia, at least in part, by dampening mucosal responses to flagellin.

A CD4+CD161+ T-Cell Subset Present in Unexposed Humans, Not Tb Patients, Are Fast Acting Cells That Inhibit the Growth of Intracellular Mycobacteria Involving CD161 Pathway, Perforin, and IFN-γ/Autophagy.

It remains undefined whether a subset of CD4+ T cells can function as fast-acting cells to control Mycobacterium tuberculosis (Mtb) infection. Here we show that the primary CD4+CD161+ T-cell subset, not CD4+CD161-, in unexposed healthy humans fast acted as unconventional T cells capable of inhibiting intracellular Mtb and BCG growth upon exposure to infected autologous and allogeneic macrophages or lung epithelial A549 cells. Such inhibition coincided with the ability of primary CD4+CD161+ T cells to rapidly express/secrete anti-TB cytokines including IFN-γ, TNF-α, IL-17, and perforin upon exposure to Mtb. Mechanistically, blockades of CD161 pathway, perforin or IFN-γ by blocking mAbs abrogated the ability of CD4+CD161+ T cells to inhibit intracellular mycobacterial growth. Pre-treatment of infected macrophages with inhibitors of autophagy also blocked the CD4+CD161+ T cell-mediated growth inhibition of mycobacteria. Furthermore, adoptive transfer of human CD4+CD161+ T cells conferred protective immunity against mycobacterial infection in SCID mice. Surprisingly, CD4+CD161+ T cells in TB patients exhibited a loss or reduction of their capabilities to produce perforin/IFN-γ and to inhibit intracellular growth of mycobacteria in infected macrophages. These immune dysfunctions were consistent with PD1/Tim3 up-regulation on CD4+CD161+ T cells in active tuberculosis patients, and the blockade of PD1/Tim3 on this subset cells enhanced the inhibition of intracellular mycobacteria survival. Thus, these findings suggest that a fast-acting primary CD4+CD161+T-cell subset in unexposed humans employs the CD161 pathway, perforin, and IFN-γ/autophagy to inhibit the growth of intracellular mycobacteria, thereby distinguishing them from the slow adaptive responses of conventional CD4+ T cells. The presence of fast-acting CD4+CD161+ T-cell that inhibit mycobacterial growth in unexposed humans but not TB patients also implicates the role of these cells in protective immunity against initial Mtb infection.

Carbonic anhydrase inhibition, antioxidant activity against alveolar epithelial cells and antibacterial effect against Klebsiella pneumoniae enabled by synthesized silica nanoparticles through laser ablation technique.

Silica (SiO2) nanoparticles (NPs) were synthesized by laser ablation method and were characterized by TEM and DLS techniques. Afterwards, their inhibition activity against carbonic anhydrase (CA) isoforms (CA I and CA II) was explored by experimental and theoretical analysis. Also, the protective effect of SiO2 NPs against H2O2-induced oxidative stress in alveolar epithelial cells (A549) were assessed by measurement of MTT, ROS level, CAT and SOD activity and GSH content. Finally, the NPs were screened for their antimicrobial activity using the MICs method against the Klebsiella pneumoniae. The result showed that the synthesized NPs have a size of around 40 nm. The inhibition activity by comparing IC50 values with acetazolamide as a positive control revealed that SiO2 NPs in comparison with acetazolamide served as potent inhibitors against CA isoforms which was also confirmed by docking studies. The cellular assays indicated that the SiO2 NPs with a concentration of 20 µg/mL stimulated a significant antioxidant activity against H2O2-induced oxidative cell damage through activation of CAT and SOD, an increase in the GSH content and reducing the level of ROS. The synthesize NPs also showed a good inhibition effect against Klebsiella pneumoniae as compared to Sulfamethoxazole as a positive control. In conclusion, this data may provide some useful information on the development of some platforms for pneumonia treatment and management.

Single-Cell Analysis of Fungal Uptake in Cultured Airway Epithelial Cells Using Differential Fluorescent Staining and Imaging Flow Cytometry.

The respiratory epithelium is the initial point of host contact for inhaled particles, leading to orchestrated, but highly heterogeneous, responses. Human airway epithelial cells (AECs) play a crucial role in host defense by promoting uptake and killing of inhaled microorganisms and concomitant cytokine production in order to recruit professional phagocytes to the site of infection. However, inhaled pathogens can also reside and replicate intracellularly to evade host immune defenses or circulating antimicrobial drugs, ultimately causing apoptosis or cell death of the infected AECs. Imaging flow cytometry (IFC) combines flow cytometry, fluorescent microscopy, and advanced data-processing algorithms to dissect the heterogeneity of the interaction of AECs and inhaled microorganisms and its outcomes at the single-cell level. Here, we describe a novel single-cell approach based on differential fluorescent staining and state-of-the-art IFC to identify, quantify, and analyze individual host-pathogen complexes from cultured AECs infected with spores of the major human fungal pathogen Aspergillus fumigatus.

Stimulation of surfactant exocytosis in primary alveolar type II cells by A. fumigatus.

Aspergillus fumigatus is an opportunistic fungal pathogen with small airborne spores (conidia) that may escape clearance by upper airways and directly impact the alveolar epithelium. Consequently, innate alveolar defense mechanisms are being activated, including professional phagocytosis by alveolar macrophages, recruitment of circulating neutrophils and probably enhanced secretion of pulmonary surfactant by the alveolar type II (AT II) cells. However, no data are available in support of the latter hypothesis. We therefore used a coculture model of GFP-Aspergillus conidia with primary rat AT II cells and studied fungal growth, cellular Ca2+ homeostasis, and pulmonary surfactant exocytosis by live cell video microscopy. We observed all stages of fungal development, including reversible attachment, binding and internalization of conidia as well as conidial swelling, formation of germ tubes and outgrowth of hyphae. In contrast to resting conidia, which did not provoke immediate cellular effects, metabolically active conidia, fungal cellular extracts (CE) and fungal culture filtrates (CF) prepared from swollen conidia caused a Ca2+-independent exocytosis. Ca2+ signals of greatly varying delays, durations and amplitudes were observed by applying CE or CF obtained from hyphae of A. fumigatus, suggesting compounds secreted by filamentous A. fumigatus that severely interfere with AT II cell Ca2+ homeostasis. The mechanisms underlying the stimulatory effects, with respect to exocytosis and Ca2+ signaling, are unclear and need to be identified.

Nicotine promotes the intracellular growth of Mycobacterium tuberculosis in epithelial cells.

Several epidemiological studies have identified the cigarette smoke as a risk factor for the infection and development of tuberculosis. Nicotine is considered the main immunomodulatory molecule of the cigarette. In the present study, we evaluated the effect of nicotine in the growth of M. tuberculosis. Lung epithelial cells and macrophages were infected with M. tuberculosis and/or treated with nicotine. The results show that nicotine increased the growth of M. tuberculosis mainly in type II pneumocytes (T2P) but not in airway basal epithelial cells nor macrophages. Further, it was observed that nicotine decreased the production of ß-defensin-2, ß-defensin-3, and the cathelicidin LL-37 in all the evaluated cells at 24 and 72 h post-infection. The modulation of the expression of antimicrobial peptides appears to be partially mediated by the nicotinic acetylcholine receptor α7 since the blockade of this receptor partially reverted the production of antimicrobial peptides. In summary, it was found that nicotine decreases the production of HBD-2, HBD-3, and LL-37 in T2P during the infection with M. tuberculosis promoting its intracellular growth.

Role of the COX2-PGE2 axis in S. pneumoniae-induced exacerbation of experimental fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease (ILD) associated with high morbidity and mortality. Patients with ILD frequently develop an acute exacerbation of their disease, which may be triggered by viral and/or bacterial infections. Prostaglandin E2 (PGE2) is an eicosanoid released in a cyclooxygenase-2 (COX2)-dependent manner and is considered to contribute to regulation of lung fibrosis. However, its role in infection-induced exacerbation of lung fibrosis is poorly defined. We found significantly increased levels of PGE2 in lung tissue of patients with ILD. Increased levels of PGE2 were also found in lung tissue of mice with AdTGF-ß1-induced lung fibrosis and even more so in Streptococcus pneumoniae exacerbated lung fibrosis. Type II alveolar epithelial cells (AT II cells) and alveolar macrophages (AM) contributed to PGE2 release during exacerbating fibrosis. Application of parecoxib to inhibit PGE2 synthesis ameliorated lung fibrosis, whereas intratracheal application of PGE2 worsened lung fibrosis in mice. Both interventions had no effect on S. pneumoniae-exacerbated lung fibrosis. Together, we found that the COX2-PGE2 axis has dual roles in fibrosis that may offset each other: PGE2 helps resolve infection/attenuate inflammation in fibrosis exacerbation but accentuates TGF-ß/AT II cell-mediated fibrosis. These data support the efficacy of COX/PGE2 interventions in the setting of non-exacerbating lung fibrosis.

A lung-on-chip model of early Mycobacterium tuberculosis infection reveals an essential role for alveolar epithelial cells in controlling bacterial growth.

We establish a murine lung-on-chip infection model and use time-lapse imaging to reveal the dynamics of host-Mycobacterium tuberculosis interactions at an air-liquid interface with a spatiotemporal resolution unattainable in animal models and to probe the direct role of pulmonary surfactant in early infection. Surfactant deficiency results in rapid and uncontrolled bacterial growth in both macrophages and alveolar epithelial cells. In contrast, under normal surfactant levels, a significant fraction of intracellular bacteria are non-growing. The surfactant-deficient phenotype is rescued by exogenous addition of surfactant replacement formulations, which have no effect on bacterial viability in the absence of host cells. Surfactant partially removes virulence-associated lipids and proteins from the bacterial cell surface. Consistent with this mechanism, the attenuation of bacteria lacking the ESX-1 secretion system is independent of surfactant levels. These findings may partly explain why smokers and elderly persons with compromised surfactant function are at increased risk of developing active tuberculosis.

Tuberculosis is a contagious respiratory disease caused by the bacterium Mycobacterium tuberculosis. Droplets in the air carry these bacteria deep into the lungs, where they cling onto and infect lung cells. Only small droplets, holding one or two bacteria, can reach the right cells, which means that just a couple of bacterial cells can trigger an infection. But people respond differently to the bacteria: some develop active and fatal forms of tuberculosis, while many show no signs of infection. With no effective tuberculosis vaccine for adults, understanding why individuals respond differently to Mycobacterium tuberculosis may help develop treatments. Different responses to Mycobacterium tuberculosis may stem from the earliest stages of infection, but these stages are difficult to study. For one thing, tracking the movements of the few bacterial cells that initiate infection is tricky. For another, studying the molecules, called 'surfactants', that the lungs produce to protect themselves from tuberculosis can prove difficult because these molecules are necessary for the lungs to inflate and deflate normally. Normally, the role of a molecule can be studied by genetically modifying an animal so it does not produce the molecule in question, which provides information as to its potential roles. Unfortunately, due to the role of surfactants in normal breathing, animals lacking them die. Therefore, to reveal the role of some of surfactants in tuberculosis, Thacker et al. used 'lung-on-chip' technology. The 'chip' (a transparent device made of a polymer compatible with biological tissues) is coated with layers of cells and has channels to simulate air and blood flow. To see what effects surfactants have on M. tuberculosis bacteria, Thacker et al. altered the levels of surfactants produced by the cells on the lung-on-chip device. Two types of mouse cells were grown on the chip: lung cells and immune cells. When cells lacked surfactants, bacteria grew rapidly on both lung and immune cells, but when surfactants were present bacteria grew much slower on both cell types, or did not grow at all. Further probing showed that the surfactants pulled out proteins and fats on the surface of M. tuberculosis that help the bacteria to infect their host, highlighting the protective role of surfactants in tuberculosis. These findings lay the foundations for a system to study respiratory infections without using animals. This will allow scientists to study the early stages of Mycobacterium tuberculosis infection, which is crucial for finding ways to manage tuberculosis.

Modelling early events in Mycobacterium bovis infection using a co-culture model of the bovine alveolus.

Bovine tuberculosis (bTB), a zoonosis mainly caused by Mycobacterium bovis has severe socio-economic consequences and impact on animal health. Host-pathogen interactions during M. bovis infection are poorly understood, especially early events which are difficult to follow in vivo. This study describes the utilisation of an in vitro co-culture model, comprising immortalised bovine alveolar type II (BATII) epithelial cells and bovine pulmonary arterial endothelial cells (BPAECs). When cultured at air-liquid interface, it was possible to follow the migration of live M. bovis Bacille Calmette-Guérin (BCG) and to observe interactions with each cell type, alongside cytokine release. Infection with BCG was shown to exert a detrimental effect primarily upon epithelial cells, with corresponding increases in IL8, TNFα, IL22 and IL17a cytokine release, quantified by ELISA. BCG infection increased expression of CD54, MHC Class I and II molecules in endothelial but not epithelial cells, which exhibited constitutive expression. The effect of peripheral blood mononuclear cell conditioned medium from vaccinated cattle upon apical-basolateral migration of BCG was examined by quantifying recovered BCG from the apical, membrane and basolateral fractions over time. The numbers of recovered BCG in each fraction were unaffected by the presence of PBMC conditioned medium, with no observable differences between vaccinated and naïve animals.

H1N1 Influenza Virus Cross-Activates Gli1 to Disrupt the Intercellular Junctions of Alveolar Epithelial Cells.

Influenza A virus (IAV) primarily infects the airway and alveolar epithelial cells and disrupts the intercellular junctions, leading to increased paracellular permeability. Although this pathological change plays a critical role in lung tissue injury and secondary infection, the molecular mechanism of IAV-induced damage to the alveolar barrier remains obscure. Here, we report that Gli1, a transcription factor in the sonic hedgehog (Shh) signaling pathway, is cross-activated by the MAP and PI3 kinase pathways in H1N1 virus (PR8)-infected A549 cells and in the lungs of H1N1 virus-infected mice. Gli1 activation induces Snail expression, which downregulates the expression of intercellular junction proteins, including E-cadherin, ZO-1, and Occludin, and increases paracellular permeability. Inhibition of the Shh pathway restores the levels of Snail and intercellular junction proteins in H1N1-infected cells. Our study suggests that Gli1 activation plays an important role in disrupting the intercellular junctions and in promoting the pathogenesis of H1N1 virus infections.

Mycobacterium tuberculosis-infected alveolar epithelial cells modulate dendritic cell function through the HIF-1α-NOS2 axis.

Tuberculosis kills more than 1 million people every year, and its control depends on the effective mechanisms of innate immunity, with or without induction of adaptive immune response. We investigated the interaction of type II alveolar epithelial cells (AEC-II) infected by Mycobacterium tuberculosis with dendritic cells (DCs). We hypothesized that the microenvironment generated by this interaction is critical for the early innate response against mycobacteria. We found that AEC-II infected by M. tuberculosis induced DC maturation, which was negatively regulated by HIF-1α-inducible NOS2 axis, and switched DC metabolism from an early and short peak of glycolysis to a low energetic status. However, the infection of DCs by M. tuberculosis up-regulated NOS2 expression and inhibited AEC-II-induced DC maturation. Our study demonstrated, for the first time, that HIF-1α-NOS2 axis plays a negative role in the maturation of DCs during M. tuberculosis infection. Such modulation might be useful for the exploitation of molecular targets to develop new therapeutic strategies against tuberculosis.

Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca2+-ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus.

Many species of pathogenic fungi deploy the unfolded protein response (UPR) to expand the folding capacity of the endoplasmic reticulum (ER) in proportion to the demand for virulence-related proteins that traffic through the secretory pathway. Although Ca2+ plays a pivotal role in ER function, the mechanism by which transcriptional upregulation of the protein folding machinery is coordinated with Ca2+ homeostasis is incompletely understood. In this study, we investigated the link between the UPR and genes encoding P-type Ca2+-ATPases in the human-pathogenic mold Aspergillus fumigatus We demonstrate that acute ER stress increases transcription of the srcA gene, encoding a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family, as well as that of pmrA, encoding a secretory pathway Ca2+-ATPase (SPCA) in the Golgi membrane. Loss of the UPR transcription factor HacA prevented the induction of srcA and pmrA transcription during ER stress, defining these ER/Golgi Ca2+ pumps as novel downstream targets of this pathway. While deletion of srcA alone caused no major deficiencies, a ΔsrcA/ΔpmrA mutant displayed a severe polarity defect, was hypersensitive to ER stress, and showed attenuated virulence. In addition, cell wall analyses revealed a striking reduction in mannose levels in the absence of both Ca2+ pumps. The ΔhacA mutant was hypersensitive to agents that block calcineurin-dependent signaling, consistent with a functional coupling between the UPR and Ca2+ homeostasis. Together, these findings demonstrate that the UPR integrates the need for increased levels of chaperone and folding enzymes with an influx of Ca2+ into the secretory pathway to support fungal growth, stress adaptation, and pathogenicity.IMPORTANCE The UPR is an intracellular signal transduction pathway that maintains homeostasis of the ER. The pathway is also tightly linked to the expression of virulence-related traits in diverse species of human-pathogenic and plant-pathogenic fungal species, including the predominant mold pathogen infecting humans, Aspergillus fumigatus Despite advances in the understanding of UPR signaling, the linkages and networks that are governed by this pathway are not well defined. In this study, we revealed that the UPR is a major driving force for stimulating Ca2+ influx at the ER and Golgi membranes and that the coupling between the UPR and Ca2+ import is important for virulence, cell wall biosynthesis, and resistance to antifungal compounds that inhibit Ca2+ signaling.