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Mtb H37Rv-infected A549 cells reduced TLR signaling activity and cytokine production of U937 cells in response to mycobacterial infection. (a) Illustration of cell culture models used for infection in this study. A549 cells were cultured on the apical surface of transwells at an air-liquid interface state for 24 hours; then the transwell insert was transferred to a well containing PMA-stimulated U937 cells for infection. The coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (b) Representative blots of immunoblotting assay for indicated components of TLR signaling cascade (left panel) and fold of changes of proteins of interest in U937 cells semiquantitatively determined by densitometric assay using ImageJ software Fiji (right panel). H37Rv-infected A549 cells showed an ability to reduce TLR signaling activity in U937 cells in response to mycobacterial infection. Concentrations of TNF-α (c), IL-10 (d), and IL-6 (e) in culture media determined by ELISA. H37Rv-infected A549 cells led a reduction of cytokine production in U937 cells in response to Mycobacteria infection. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗∗p<0.01; compared to infection of U937 cell alone, ΔΔp<0.01. NI: noninfected control; AI: infection was performed on A549 cell alone; UI: infection was performed on U937 alone; CI: infection was performed on both A549 cells and U937 cells.
Source publication
Both alveolar macrophages (AMs) and alveolar epithelial cells (AECs) are main targets of Mycobacterium tuberculosis ( M. tuberculosis ( Mtb )). Intercellular communications between mucosal AECs and AMs have important implications in cellular responses to exogenous insults. However, molecular mechanisms underpinning interactions responding to Mtb re...
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Citations
... Although the constitutive activation events for PI3K/Akt/mTOR and TLR signaling are different, these signalling networks are related and susceptible to complicated cross-talk and feedback interactions [42]. Altogether, the applied combinational therapy with R848 and BEZ235 showed more effective results than the conventional chemotherapy in murine AML model of the current study. ...
Keywords: Acute myeloid leukemia Combinational therapy BEZ235 PI3K/mTOR R848 TLR7/8 A B S T R A C T Background: Due to the high relapse rate and toxicity of the common therapies in patients with acute myeloid leukemia (AML), modifications in the treatment strategies are required. The present study was conducted to determine the effects of combinational therapy with a dual PI3K/mTOR inhibitor, BEZ235, and TLR7/8 agonist, R848, on murine AML model. Methods: BEZ235 and R848 were administered to AML leukemic mice in either a single or combination treatment. Frequency of T-CD4 + , T-CD8 + , MDSCs, NK, exhausted T cells and the degranulation levels was measured via flow cytometry. The cytotoxicity and proliferation levels were evaluated by MTT assay. Then, the expression of iNOS, arginase-1, PD-L1, Gal-9, PVR, IFN-γ, TNF-α, IL-4, IL-10, IL-12 and IL-17 was investigated by Real-Time PCR. Organomegaly, body weight and survival rate were also monitored. Results: Following combinational therapy with BEZ235 and R848, increasing in the frequency of anti-tumor immune cells including T-CD4 + cells and M1 macroghages, and decreasing in pro-tumor immune cells including MDSCs, exhausted T-CD4 + and T-CD8 + cells and also M2 macrophages were observed. The functional defects of immune cells in term of proliferation, cytotoxicity, degranulation, and cytokines expression were improved in leukemic mice after treatment with BEZ235 and R848. Finally, organomegaly, body weight and survival analysis showed significant improvements after treatment with BEZ235 and R848. Conclusion: Taken together, we indicated that the combinational therapy with BEZ235 and R848 could be considered as a potential and powerful therapeutic option for AML patients. Further clinical studies are required to expand our current findings.
... LPS interacts with TLR4, triggering the production of inflammatory mediators via induction of the AKT/NFκB axis (Ojaniemi et al. 2003;Su et al. 2017). In addition, a recent study showed that the TLR-driven PI3K/AKT/mTOR-dependent inflammatory response of U937 macrophage-like cells is inhibited by A549 epithelial cells due to a reduction in the production of soluble cytokines and mediators upon infection with Mycobacterium tuberculosis (Yang et al. 2018). ...
Macrophages display an array of activation phenotypes depending on the activation signal and the cellular microenvironment. The type and magnitude of the response depend on signaling molecules as well as on the epigenetic and metabolic status of the cells at the time of activation. The AKT family of kinases consists of three isoforms encoded by independent genes possessing similar functions and structures. Generation of research tools such as isoform-specific gene deletion mutant mice and cells and isoform-specific antibodies has allowed us to understand the role of each kinase isoform in macrophage activation and homeostasis. This chapter discusses the current evidence on the role of AKT kinases in macrophage activation, polarization, and homeostasis, highlighting the gaps in knowledge and future challenges in the field.
... According to KEGG pathway prediction, 14 common pathways are shared by these two candidate miRNAs. Among them, the MAPK, mTOR, and PI3K-Akt signaling pathways have been found to be involved in the host response to TB disease (20)(21)(22)(23). Matrix metalloproteinases (MMPs), downstream effector molecules of the MAPK signaling pathway, induce lung tissue remodeling and contribute to early TB granuloma formation (24,25). ...
... Additionally, a variety of MMP inhibitors have been used to study the immunomodulatory effects of MMPs in M.tb infection, and have shown the immunomodulatory roles of MMPs in M.tb pathogenesis (26,27). PI3K/Akt/mTOR signaling was reported to be involved in the epithelial cell-modulated, M.tb-activated Toll-like receptor signaling pathway, and observational study provided evidence of an association between mTOR polymorphism and TB susceptibility (22,23). Although no common target gene was found for the two miRNAs, MAP2k4 and MAP2k1 from hsa-miR-16-5p and MAP3k1 from hsa-miR-451a were found, further indicating the importance of the MAPK pathway in TB development. ...
Approximately a quarter of the world population are infected with M. tuberculosis and about 5% to 10% of these might develop active disease in their lifetime. Preventive treatment could effectively protect individuals at a high risk of developing active disease from LTBI, and is regarded as a critical component of End TB Strategies.
... According to GO and KEGG analysis, the genes were enriched in the PI3K-Akt signaling pathway, the proteoglycan pathway in cancer, and Ras signaling. Yang et al. showed that the inflammatory response of macrophage-like cells to MTB can be attenuated by modulating the PI3K/Akt/mTOR signaling pathway [38]. Other studies have shown that PI3K/ AKT/mTOR signaling pathways are suppressed in patients with active pulmonary TB [39]. ...
Pulmonary tuberculosis (TB) is a chronic infectious disease that is caused by respiratory infections, principally Mycobacterium tuberculosis. Increasingly, studies have shown that circular (circ)RNAs play regulatory roles in different diseases through different mechanisms. However, their roles and potential regulatory mechanisms in pulmonary TB remain unclear. In this study, we analyzed circRNA sequencing data from adjacent normal and diseased tissues from pulmonary TB patients and analyzed the differentially expressed genes. We then constructed machine learning models and used single-factor analysis to identify hub circRNAs. We downloaded the pulmonary TB micro (mi)RNA (GSE29190) and mRNA (GSE83456) gene expression datasets from the Gene Expression Omnibus database and performed differential expression analysis to determine the differentially expressed miRNAs and mRNAs. We also constructed a circRNA–miRNA–mRNA interaction network using Cytoscape. Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were used to predict the biological functions of the identified RNAs and determine hub genes. Then, the STRING database and cytoHubba were used to construct protein-protein interaction networks. The results showed 125 differentially expressed circRNAs in the adjacent normal and diseased tissues of pulmonary TB patients. Among them, we identified three hub genes associated with the development of pulmonary TB: hsa_circ_0007919 (upregulated), hsa_circ_0002419 (downregulated), and hsa_circ_0005521 (downregulated). Through further screening, we determined 16 mRNAs of potential downstream genes for hsa-miR-409-5p and hsa_circ_0005521 and established an interaction network. This network may have important roles in the occurrence and development of pulmonary TB. We constructed a model with 100% prediction accuracy by machine learning and single-factor analysis. We constructed a protein-protein interaction network among the top 50 hub mRNAs, with FBXW7 scoring the highest and SOCS3 the second highest. These results may provide a new reference for the identification of candidate markers for the early diagnosis and treatment of pulmonary TB.
... Our work together with previous studies suggests that the treatment based on TLR agonists alone might not always be efficient as a monotherapy, and hence comes the need for introducing combinatorial treatment (49,53). Indeed, although the constitutive activation events for TLR and PI3K/Akt/mTOR signaling are independent, these signaling networks are closely related and are subject to complex cross-talk and feedback interactions (54). The combinational treatment with R848 and BEZ235 can further reduce the growth inhibition, and the expression of immune checkpoint ligands via the down-regulation of STAT3 expression and phosphorylation in WEHI-3 cells. ...
Background:
Several PI3K/Akt/mTOR pathway inhibitors and TLR agonists induce tumor cell death. However, the mechanisms of these therapeutic approaches in acute myeloid leukemia (AML) cells are still unknown.
Objectives:
To investigate the effects of BEZ235, as a dual inhibitor of PI3K and mTOR pathways, and TLR7/8 agonist R848 on the expression and regulation of the immune inhibitory molecules in myeloid leukemia cells.
Methods:
WEHI-3 leukemia cells were incubated with dual PI3K and mTOR inhibitor BEZ235 and TLR7/8 agonist R848 for 48 hrs. Firstly, cell viability was assessed by MTT method. The semi-quantitative relative mRNA expression of Galectin-9 (Gal-9), PD-L1, PVR, and STAT3 was assessed according to HPRT as a housekeeping gene. Finally, the protein expression of phosphorylated STAT3 was evaluated by western blotting analysis.
Results:
WEHI-3 cells showed growth inhibition following treatment with BEZ235 and R848 whose combination exerted more proliferation arrest. The mRNA expression of Gal-9, PD-L1 and PVR immune checkpoint molecules significantly reduced in treated cells with BEZ235 and R848. Combined treatment indicated more reduction compared with the single treatment. Finally, the expression and phosphorylation of STAT3 were down-regulated after a single or dual treatment with BEZ235 and R848.
Conclusion:
Our results conclude that treatment with the combination of BEZ235 and R848 interferes with immune evasion mechanisms through STAT3-signaling pathway in WEHI-3 leukemia cells.
... Culture of the monocyte cell line U937 in presence of Mtb-infected A549 cells resulted in reduced Mtb-triggered TLR signalling in the monocytes, compared with infected monocytes alone. 43 This suggests that EC influence the inflammatory response of immune cells. Additionally, submerged PBEC cultures produced anti-inflammatory cytokines IL-10 and IL-22 in response to BCG infection. ...
... Alternatively, Mtb-infected macrophages may be introduced to the basal compartment of Transwell systems. 43 While such methods do illustrate the multifunctionality of the ALI model, they bypass the epithelium as a first point of hostpathogen contact. Airway ALI models have been successfully infected with various bacteria, fungi and viruses, suggesting that bronchial EC are less susceptible to Mtb infection in comparison to other pathogens. ...
The lung epithelium has long been overlooked as a key player in tuberculosis disease. In addition to acting as a direct barrier to Mycobacterium tuberculosis (Mtb), epithelial cells (EC) of the airways and alveoli act as first responders during Mtb infections; they directly sense and respond to Mtb by producing mediators such as cytokines, chemokines and antimicrobials. Interactions of EC with innate and adaptive immune cells further shape the immune response against Mtb. These three essential components, epithelium, immune cells and Mtb, are rarely studied in conjunction, owing in part to difficulties in coculturing them. Recent advances in cell culture technologies offer the opportunity to model the lung microenvironment more closely. Herein, we discuss the interplay between lung EC, immune cells and Mtb and argue that modelling these interactions is of key importance to unravel early events during Mtb infection.
... In addition, the TLR signaling may also induce autophagy: mTOR senses intracellular energy levels and regulates metabolism through the RTK-PI3K-AKT-mTOR pathway, and if such a pathway is blocked, autophagy is to take place [137,138]. This was corroborated by Yang and his colleagues, who found that in A549 cells, TLR-MyD88-TRAF6 signaling induces the activation of the PI3K-AKT-mTOR pathway, thus indirectly inhibited autophagy and promoted the development of inflammation [139]. Among the many NLRP3 signaling pathways, the cross-talking between AMPK, mTORC, and autophagy is commonly observed. ...
The NLRP3 inflammasome is a multi-molecular complex that acts as a molecular platform to mediate caspase-1 activation, leading to IL-1β/IL-18 maturation and release in cells stimulated by various pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). This inflammasome plays an important role in the innate immunity as its activation can further promote the occurrence of inflammation, enhance the ability of host to remove pathogens, and thus facilitate the repair of injured tissues. But if the inflammasome activation is dysregulated, it will cause the development of various inflammatory diseases and metabolic disorders. Therefore, under normal conditions, the activation of inflammasome is tightly regulated by various positive and negative signaling pathways to respond to the stimuli without damaging the host itself while maintaining homeostasis. In this review, we summarize recent advances in the major signaling pathways (including TLRs, MAPK, mTOR, autophagy, PKA, AMPK, and IFNR) that regulate NLRP3 inflammasome activation, providing a brief view of the molecular network that regulates this inflammasome as a theoretical basis for therapeutic intervention of NLRP3 dysregulation-related diseases.
... Figure 7 illustrates this using the example of SNPs within and proximal to gene loci associated with PI3K/ AKT signalling, which based on IPA data mining for bAM DE genes was a highly activated pathway, particularly at 24 hpi. In this regard, previous work has shown that macrophage PI3K/AKT signalling is key to a range of cellular processes associated with host-pathogen interaction in MTBC infections, including modulation of cell death pathways, manipulation of signalling downstream of TLRs, and initiation of granuloma formation [79][80][81][82][83][84]. We have also recently demonstrated that genes encoding protein products embedded in the PI3K/AKT pathway are primary targets for chromatin modifications that substantially alter bAM gene expression in response to M. bovis infection [46]. ...
... It is noteworthy, therefore, that our analysis prioritised SNPs at the TNF gene locus, a key nexus for host-pathogen interaction in mycobacterial infections. In addition, another important component of host-pathogen interaction and target for immunoevasion by intracellular mycobacterial infections is the PI3K/AKT pathway [33,79,83] and our integrative genomics approach was able to prioritise genomic variation at genes encoding ligands and receptors within this signalling network, specifically TNXB, GNG11, ITGB3, and VWF (see Fig. 7). ...
Background
Bovine TB (bTB), caused by infection with Mycobacterium bovis , is a major endemic disease affecting global cattle production. The key innate immune cell that first encounters the pathogen is the alveolar macrophage, previously shown to be substantially reprogrammed during intracellular infection by the pathogen. Here we use differential expression, and correlation- and interaction-based network approaches to analyse the host response to infection with M. bovis at the transcriptome level to identify core infection response pathways and gene modules. These outputs were then integrated with genome-wide association study (GWAS) data sets to enhance detection of genomic variants for susceptibility/resistance to M. bovis infection.
Results
The host gene expression data consisted of RNA-seq data from bovine alveolar macrophages (bAM) infected with M. bovis at 24 and 48 h post-infection (hpi) compared to non-infected control bAM. These RNA-seq data were analysed using three distinct computational pipelines to produce six separate gene sets: 1) DE genes filtered using stringent fold-change and P -value thresholds (DEG-24: 378 genes, DEG-48: 390 genes); 2) genes obtained from expression correlation networks (CON-24: 460 genes, CON-48: 416 genes); and 3) genes obtained from differential expression networks (DEN-24: 339 genes, DEN-48: 495 genes). These six gene sets were integrated with three bTB breed GWAS data sets by employing a new genomics data integration tool— gwinteR . Using GWAS summary statistics, this methodology enabled detection of 36, 102 and 921 prioritised SNPs for Charolais, Limousin and Holstein-Friesian, respectively.
Conclusions
The results from the three parallel analyses showed that the three computational approaches could identify genes significantly enriched for SNPs associated with susceptibility/resistance to M. bovis infection. Results indicate distinct and significant overlap in SNP discovery, demonstrating that network-based integration of biologically relevant transcriptomics data can leverage substantial additional information from GWAS data sets. These analyses also demonstrated significant differences among breeds, with the Holstein-Friesian breed GWAS proving most useful for prioritising SNPS through data integration. Because the functional genomics data were generated using bAM from this population, this suggests that the genomic architecture of bTB resilience traits may be more breed-specific than previously assumed.
... Figure 6 illustrates this using the example of SNPs within and proximal to gene loci associated with PI3K/AKT signalling, which based on IPA data mining for bAM DE genes was a highly activated pathway, particularly at 24 hpi. In this regard, previous work has shown that macrophage PI3K/AKT signalling is key to a range of cellular processes associated with host-pathogen interaction in MTBC infections, including modulation of cell death pathways, manipulation of signalling downstream of TLRs, and initiation of granuloma formation [76][77][78][79][80][81]. We have also recently demonstrated that genes encoding protein products embedded in the PI3K/AKT pathway are primary targets for chromatin modi cations that substantially alter bAM gene expression in response to M. bovis infection [46]. ...
Background: Bovine TB (BTB), caused by infection with Mycobacterium bovis, is a major endemic disease affecting global cattle production, particularly in many developing countries. The key innate immune that first encounters the pathogen is the alveolar macrophage, previously shown to be substantially reprogrammed during intracellular infection by the pathogen. Here we use differential expression, and correlation- and interaction-based network approaches to analyse the host response to infection with M. bovis at the transcriptome level to identify core infection response pathways and gene modules. These outputs were then integrated with genome-wide association study (GWAS) data sets to enhance detection of genomic variants for susceptibility/resistance to M. bovis infection. Results: The host gene expression data consisted of bovine RNA-seq data from alveolar macrophages infected with M. bovis at 24 and 48 hours post-infection. These RNA-seq data were analysed using three distinct analysis pipelines and novel response pathways and modules were further refined using cross-comparison and integration of the results. First, a differential expression analysis was carried out to determine the most significantly differentially expressed (DE) genes between conditions at each time point. Second, two networks were constructed at each time point using gene correlation patterns to determine changes in expression across conditions. Functional sub-modules within each correlation network were selected by statistical criteria for modularity. Third, a base gene interaction network of the mammalian host response to mycobacterial infection was generated using the GeneCards database and InnateDB. Differential gene expression data were superimposed on this base network to extract functional modules of interconnected DE genes. Conclusions: Bovine GWAS data was obtained from a published BTB susceptibility/resistance study. The results from the three parallel analyses were integrated with this data to determine which of the three approaches identified genes significantly enriched for SNPs associated with susceptibility/resistance to M. bovis infection. Results indicate distinct and significant overlap in SNP discovery, demonstrating that network-based integration of biologically relevant transcriptomics data can leverage substantial additional information from GWAS data sets.
Akt is an important kinase in metabolism. Akt also phosphorylates and activates endothelial and neuronal nitric oxide (NO) synthases (eNOS and nNOS, respectively) expressed in M0 (unpolarized) macrophages. We showed that e/nNOS NO production downstream of bitter taste receptors enhances macrophage phagocytosis. In airway epithelial cells, we also showed that the activation of Akt by a small molecule (SC79) enhances NO production and increases levels of nuclear Nrf2, which reduces IL-8 transcription during concomitant stimulation with Toll-like receptor (TLR) 5 agonist flagellin. We hypothesized that SC79’s production of NO in macrophages might likewise enhance phagocytosis and reduce the transcription of some pro-inflammatory cytokines. Using live cell imaging of fluorescent biosensors and indicator dyes, we found that SC79 induces Akt activation, NO production, and downstream cGMP production in primary human M0 macrophages. This was accompanied by a reduction in IL-6, IL-8, and IL-12 production during concomitant stimulation with bacterial lipopolysaccharide, an agonist of pattern recognition receptors including TLR4. Pharmacological inhibitors suggested that this effect was dependent on Akt and Nrf2. Together, these data suggest that several macrophage immune pathways are regulated by SC79 via Akt. A small-molecule Akt activator may be useful in some infection settings, warranting future in vivo studies.
