Cellular Microbiology

Objective. Smoking is a primary hazard factor for chronic obstructive pulmonary disease (COPD), which induced a decrease in intestinal Akkermansia muciniphila abundance and Th17 imbalance in COPD. This study analyzed the changes of gut microbiota metabolism and Akkermansia abundance in patients with smoking-related COPD and explored the potential function of Akkermansia muciniphila in smoke-induced COPD mice. Methods. Gut microbiota diversity and metabolic profile were analyzed by 16S rRNA sequence and metabolomics in COPD patients. The IL-1β, IL-17, TNF-α, and IL-6 levels were tested by ELISA. Lung tissue damage was observed by HE staining. The expression of cleave-caspase 3, trophoblast antigen 2 (TROP2), and LC3 in lung tissues were analyzed by IHC or IF. The p-mTOR, mTOR, p62, and LC3 expression in lung tissues were tested by western blot. Results. The levels of IL-17, IL-1β, TNF-α, and IL-6 in the peripheral blood of COPD patients increased significantly. The number and alpha diversity of gut microbiota were decreased in COPD patients. The abundance of Akkermansia muciniphila in gut of COPD patients was decreased, and the metabolic phenotype and retinol metabolism were changed. In the retinol metabolism, the retinol and retinal were significantly changed. Akkermansia muciniphila could improve the alveolar structure and inflammatory cell infiltration in lung tissue, reduce the IL-17, TNF-α, and IL-6 levels in peripheral blood, promote the p-mTOR expression, and inhibit the expression of autophagy-related proteins in smoke-induced COPD mice. Conclusion. The number and alpha diversity of gut microbiota were decreased in patients with smoking-related COPD, accompanied by decreased abundance of Akkermansia muciniphila, and altered retinol metabolism function. Gut Akkermansia muciniphila ameliorated lung injury in smoke-induced COPD mice by inflammation and autophagy.

The influenza virus induces cellular apoptosis during viral propagation, and controlling this virus-induced apoptosis process has been shown to have significant antiviral effects. The proapoptotic BH3-only protein Noxa is a strong inducer of apoptosis that can be activated by this virus, suggesting that Noxa has the potential as an anti-influenza target. To assess the value of Noxa as an antiviral target, we utilized CRISPR/Cas9 technology to produce a Noxa-knockout cell line. We found that the knockout of Noxa resulted in a dramatic reduction in the cytopathic effect induced by the influenza virus. Moreover, Noxa knockout decreased the expression of influenza viral proteins (NP, M2, HA, and NS2). In addition, Noxa deficiency triggered a complete autophagic flux to weaken influenza virus-induced autophagosome accumulation, indicating that Noxa may be a promising antiviral target for controlling influenza virus infections.

Mycobacterium avium subsp. hominissuis (M. avium) is an opportunistic environmental pathogen that typically infects patients with existing lung conditions such as cystic fibrosis or COPD. Pulmonary M. avium infection generates peribronchial granulomas that contain infected macrophages and multinucleated giant cells (MGCs). While granuloma formation with MGCs is a common feature of mycobacterial infection, the role of MGCs within the granulomas as well as in the host-pathogen interaction is poorly understood. To shed light on the role of MGCs, we established a novel in vitro model utilizing THP-1 cells stimulated with a combination of IFN-γ and TNF-α. In this study, we show that MGCs can take up M. avium, which replicates intracellularly before leaving the cell. Bacteria that escape the MGC exhibit a highly invasive phenotype, which warrants further evaluation. Characterization of MGCs with transmission electron microscopy revealed an accumulation of cytoplasmic lipid droplets, autophagic activity, and multiple nuclei. Autophagy markers are upregulated in both uninfected and infected MGCs early in infection, measured by RT-qPCR analysis of Beclin-1 and LC3. Inhibition of autophagy with siRNA significantly reduced M. avium survival significantly in THP-1 macrophages. Depletion of host cholesterol and sphingomyelin in MGCs also resulted in decreased survival of M. avium. These processes potentially contribute to the formation of a supportive intracellular environment for the pathogen. Collectively, our results suggest that M. avium is adapted to replicate in MGCs and utilize them as a springboard for local spread.

Objectives. The effects of Kluyveromyces marxianus on high-fat diet- (HFD-) induced kidney injury (KI) were explored. Methods. HFD-induced KI model was established using male C57BL/6 mice and treated with K. marxianus JLU-1016 and acid-resistant (AR) strain JLU-1016A. Glucose tolerance was evaluated via an oral glucose tolerance test (OGTT). KI was measured using Hematoxylin and Eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. The chemical indexes were analyzed, including lipid profiles, inflammatory cytokines, and creatinine. The levels of Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) or phospho-NF-κB p65 (Ser536) and alpha inhibitor of NF-κB (IκBα) were measured using qPCR and Western blot. The gut microbiota was sequenced using high-throughput sequencing. Results. HFD induction increased OGTT value, KI severity, oxidative stress, inflammatory cytokines, oxidative stress, apoptotic rate, creatinine levels, and the expression of TLR4/NF-κB, phospho-NF-κB p65 (Ser536), and IκBα deteriorated lipid profiles () and reduced gut microbiota abundance. K. marxianus treatment ameliorated HFD-induced metabolic disorders and reversed these parameters (). Compared with the control, HFD induction increased the proportion of Firmicutes but reduced the proportion of Bacteroidetes and Lactobacillus. K. marxianus JLU-1016 and AR strain JLU-1016A treatments improved gut microbiota by reducing the proportion of Firmicutes and increasing the proportion of Bacteroidetes and Lactobacillus in the KI model (). Helicobacter has been identified with many infectious diseases and was increased after HFD induction and inhibited after K. marxianus JLU-1016 and AR strain JLU-1016A treatments. The strain JLU-1016A exhibited better results possibly with acid-tolerance properties to pass through an acidic environment of the stomach. Conclusions. K. marxianus may have a beneficial effect on KI by improving gut microbiota and inhibiting TLR4/NF-κB pathway activation.

In clinical practice, urinary tract infections (UTIs) are second only to respiratory infections in terms of infectious diseases. In recent years, drug resistance of Escherichia coli (E. coli) has increased significantly. The therapeutic effects of Shionone on UTI were assessed by modelling UTI in SD rats and SV-HUC-1 cells with E. coli solution. After treatment of Shionone, the UTI rat model showed a decrease in wet weight/body weight of bladder, as well as a reduction in cellular inflammatory infiltration of bladder tissue and a decrease in urinary levels of IL-6, IL-1β, and TNF-α. In addition, the levels of proinflammatory factors were significantly reduced in a dose-dependent manner in UTI cell model treated with different doses of Shionone (5, 10, and 20 μg/kg). The results of immunofluorescence analysis in both in vivo and in vitro experiments revealed that Shionone reduced bacterial load and the number of E. coli colonies growing on the plates was greatly reduced. These results suggested that Shionone has a good therapeutic effect on UTI, achieved by reducing bacterial load in bladder epithelial cells. The data presented here provide a basis for further research into the treatment of UTI.

The tumor and tissue microbiota of human beings have recently been investigated. Gut permeability is known as a possible resource for the positive detection of tissue bacteria. Herein, we report that microbiota were detected in high abundance in the hepatocytes of healthy rats and that they were shared with the gut microbiota to an extent. We assessed male Sprague Dawley (SD) rats for the 16S ribosomal ribonucleic acid (rRNA) gene. After the rats were sacrificed by blood drainage from the portal vein, we extracted total deoxyribonucleic acid (DNA) from their ileal and colonic contents and liver tissues. The V3–V4 region of the 16S rRNA gene was amplified by polymerase chain reaction (PCR) and sequenced using an Illumina HiSeq 2500 platform. Sequences were assigned taxonomically by the SILVA database. We also detected bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA) in situ using immunofluorescence (IF) and western blotting and the 16S rRNA gene using fluorescent in situ hybridization (FISH). In the livers of six rats, we detected effective tags of the 16S rRNA gene and clustered them into 1003 kinds of operational taxonomic units (OTUs; , 729–893). Rats showed conservation of bacterial richness, abundance, and evenness. LPS and the 16S rRNA gene were detected in the nuclei of hepatocytes. The main function composition of the genomes of annotated bacteria was correlated with metabolism (). Gram negativity was about 1.6 times higher than gram positivity. The liver microbiome was shared with both the small and large intestines but showed significantly higher richness and evenness than the gut microbiome, and the β-diversity results showed that the liver microbiome exhibited significantly higher similarity than the small and large intestines (). Our results suggest that the bacteria in the liver microbiome are hidden intracellular inhabitants in healthy rat livers.