Activation of Nrf2 modulates protective immunity against Mycobacterium tuberculosis infection in THP1-derived macrophages.

Tuberculosis (TB), caused by mycobacterium tuberculosis (M. tuberculosis) infection, is one of the leading causes of death globally and poses a threat to public health. During infection, M. tuberculosis causes redox imbalance and dysfunctions of protective immunity. Transcription factor nuclear factor erythroid 2 (NF-E2)-related factor (Nrf2) is a major modulator of cellular redox homeostasis via transcriptional induction of cytoprotective genes to protect cell against the damage from insults. Thus, we hypothesize that Nrf2 may regulate protective immunity against M. tuberculosis. RNA-seq and immunoblotting results suggested that the expression of Nrf2 protein increased after M. tuberculosis infection, and decreased upon long-term M. tuberculosis infection, while Keap1 protein maintained a low expression level during M. tuberculosis infection. Furthermore, Nrf2 activator sulforaphane (SFN) decreased proinflammatory cytokines production, phagocytosis and host cell apoptosis, while increasing ROS levels and promoting autophagy in THP1 macrophages infected with M. tuberculosis. In addition, SFN-activated Nrf2 augmented bacterial killing by macrophages, which might be due to the regulation of protective immunity via Nrf2. Combined, our results extend the understanding of the complex innate immunity regulation by Nrf2 against mycobacterial infection. Also, these findings suggested that the regulation of Nrf2 signaling cascade could be used as a therapeutic target for the treatment of TB patients and the development of better anti-TB vaccines.

Trained immunity contributes to the prevention of Mycobacterium tuberculosis infection, a novel role of autophagy.

Mycobacterium tuberculosis (M. tuberculosis) is the pathogen which causes tuberculosis (TB), a significant human public health threat. Co-infection of M. tuberculosis and the human immunodeficiency virus (HIV), emergence of drug resistant M. tuberculosis, and failure to develop highly effective TB vaccines have limited control of the TB epidemic. Trained immunity is an enhanced innate immune response which functions independently of the adaptive/acquired immune system and responds non-specifically to reinfection with invading agents. Recently, several studies have found trained immunity has the capability to control and eliminate M. tuberculosis infection. Over the past decades, however, the consensus was adaptive immunity is the only protective mechanism by which hosts inhibit M. tuberculosis growth. Furthermore, autophagy plays an essential role in the development of trained immunity. Further investigation of trained immunity, M. tuberculosis infection, and the role of autophagy in this process provide new possibilities for vaccine development. In this review, we present the general characteristics of trained immunity and autophagy. We additionally summarize several examples where initiation of trained immunity contributes to the prevention of M. tuberculosis infection and propose future directions for research in this area.

Peripheral blood non-canonical small non-coding RNAs as novel biomarkers in lung cancer.

One unmet challenge in lung cancer diagnosis is to accurately differentiate lung cancer from other lung diseases with similar clinical symptoms and radiological features, such as pulmonary tuberculosis (TB). To identify reliable biomarkers for lung cancer screening, we leverage the recently discovered non-canonical small non-coding RNAs (i.e., tRNA-derived small RNAs [tsRNAs], rRNA-derived small RNAs [rsRNAs], and YRNA-derived small RNAs [ysRNAs]) in human peripheral blood mononuclear cells and develop a molecular signature composed of distinct ts/rs/ysRNAs (TRY-RNA). Our TRY-RNA signature precisely discriminates between control, lung cancer, and pulmonary TB subjects in both the discovery and validation cohorts and outperforms microRNA-based biomarkers, which bears the diagnostic potential for lung cancer screening.

LPS restores protective immunity in macrophages against Mycobacterium tuberculosis via autophagy.

Autophagy has been identified as an important immune regulatory mechanism. Recent studies have linked macrophage autophagy with innate immune responses against Mycobacterium tuberculosis (M. tuberculosis), which can survive within macrophages by blocking fusion of the phagosome with lysosomes. These findings suggest that autophagy is a regulatable cellular mechanism of M. tuberculosis defense in macrophages. Transcriptomic profiles in human blood in TB patients suggest that M. tuberculosis affects autophagy related pathways. In order to better understand the role of macrophage autophagy in enhancing protective immunity against M. tuberculosis, in this study, we investigate the effects of the autophagy activators rapamycin and LPS in macrophage autophagy and immunity against M. tuberculosis. We confirm that rapamycin and LPS induce autophagy in M. tuberculosis infected THP-1-derived macrophages or PMA primed THP-1 macrophages [THP-1(A)]. LPS restores M. tuberculosis-inhibited IL-12 synthesis and secretion in THP-1(A) cells via autophagy. Similarly, autophagy activators increase IL-12 synthesis and secretion in THP-1(A) cells. These studies demonstrate the importance of autophagy in M. tuberculosis elimination in macrophages and may lead to novel therapies for tuberculosis and other bacterial infections.

Dysregulation of the miR-325-3p/DPAGT1 axis supports HBV-positive HCC chemoresistance.

BACKGROUND: Chemotherapeutic resistance in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC) patients is an unfortunate side effect of standard chemotherapy. This situation necessitates a better understanding of the molecular pathways underlying HBV + HCC chemoresistance in order to aid the development of novel chemotherapeutic targets. METHODS: We generated two doxorubicin (DOX)-resistant HBV + HCC sublines HepG2.2.15 and Huh7-1.3. qRT-PCR was used to evaluate dysregulation in hexosamine pathway genes in chemosensitive and chemoresistant HBV + HCC cell lines in vitro. Western blots, luciferase reporter assays, and in vivo xenograft tumor studies were conducted to reveal the role of the miRNA-325-3p/DPAGT1 axis in HBV + HCC chemoresistance. RESULTS: The hexosamine pathway gene dolichyl-phosphate N-acetylglucosamine phosphotransferase 1 (DPAGT1) was found to be upregulated in both DOX-resistant cell lines. Enhancing DPAGT1 activity significantly improved the survival of DOX-resistant cells. Silencing or pharmacological inhibition of DPAGT1 inhibited xenograft tumor growth under DOX-treated conditions. DPAGT1 upregulation was associated with higher levels of stemness-related markers and ATP-binding cassette (ABC) drug efflux transporters in DOX-resistant cell lines. miR-325-3p was found to negatively modulate DPAGT1 expression and phenocopied the effects of DPAGT1 silencing in vitro and in vivo. In HBV + HCC patients treated with transarterial chemoembolization (TACE), high and low levels of tumor DPAGT1 and miR-325-3p expression, respectively, were associated with a poor chemotherapeutic response. CONCLUSIONS: Our findings provide novel insights into the role of miR-325-3p/DPAGT1 axis dysregulation in supporting HBV + HCC chemoresistance.

Hydrogen peroxide promotes the activation of preeclampsia peripheral T cells.

Preeclampsia (PE) is a pregnancy disorder with a high mortality rate. Patients with PE exhibit systemic high oxidative stress status and inflammatory immune activation. This study aims to define the role of H2O2 in the activation of neutrophils and T lymphocytes in PE patients. CD3+/HLA-DR+ cells in blood from PE patients are remarkably increased compared with those of normal non-pregnancies or normal pregnancies, while the percentage of CD3+/CD62L+ cells is significantly reduced in PE patients compared to normal pregnancies. Furthermore, CD62L levels in granulocytes of periphery blood of PE patients are significantly higher than non-pregnancies, but significantly lower than normal pregnancies. To characterize the effects of intracellular reactive oxygen species (ROS) on T lymphocyte activation in PE patients, PBMCs from normal pregnancies were challenged with H2O2, and intracellular ROS levels in neutrophil granulocytes, as well as T cell surface marker levels, have been determined. We confirm that H2O2 exposure increases intracellular ROS levels in neutrophil granulocytes, and increases the proportion of CD3+/HLA-DR+ cells, but does not alter the percentage of CD3+/CD62L+ cells in PBMCs. Our study has confirmed dysregulated CD3+/HLA-DR+ and CD3+/CD62L+ T lymphocytes in PE patient peripheral blood, and the dysregulative effects of H2O2 on T lymphocyte activation, suggesting a novel mechanism of immune activation in PE.

Quantitative assessment of HLA-DQ gene polymorphisms with the development of hepatitis B virus infection, clearance, liver cirrhosis, and hepatocellular carcinoma.

Hepatitis B is one of the most common infectious diseases, which leads to public health problems in the world, especially in Asian counties. In recent years, extensive human genetic association studies have been carried out to identify susceptible genes and genetic polymorphisms to understand the genetic contributions to the disease progression of HBV infection. HLA-DQ gene variations have been reported to be associated with HBV infection/clearance, disease progression and the development of hepatitis B-related complications, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC). However, the results are either inconclusive or controversial. Therefore, to derive a more precise estimation of the association, a meta-analysis was performed. Our data revealed that the HLA-DQ alleles rs2856718-G, rs7453920-A and rs9275319-G were significantly associated with decreased risk of HBV infection and HBV natural clearance. Logistic regression analyses showed that HLA-DQ alleles rs9275572-A significantly increased HBV infection clearance, and decreased HBV natural clearance. However, rs2856718-G and rs9275572-A were not associated with development of cirrhosis. The HLA-DQ polymorphisms (rs2856718 and rs9275572) were associated with a decreased HBV-related HCC risk in all genetic models, but rs9272105-A increased the risk of HBV-related HCC. In addition, no significant association was observed between HLA-DQ rs9275319-G polymorphism and HBV-related HCC. These stratified analyses were limited due to relatively modest size of correlational studies. In future, further investigation on a large population and different ethnicities are warranted. Our findings contribute to the personalized care and prognosis in hepatitis B.

Potential Diagnostic Power of Blood Circular RNA Expression in Active Pulmonary Tuberculosis.

BACKGROUND: Circular RNAs (circRNAs) are a class of novel RNAs with important biological functions, and aberrant expression of circRNAs has been implicated in human diseases. However, the feasibility of using blood circRNAs as disease biomarkers is largely unknown. METHODS: We explored the potential of using human peripheral blood mononuclear cell (PBMC) circRNAs as marker molecules to diagnose active pulmonary tuberculosis (TB). FINDINGS: First, we demonstrated that circRNAs are widely expressed in human PBMCs and that many are abundant enough to be detected. Second, we found that the magnitude of PBMC circRNAs in TB patients was higher than that in the paired healthy controls. Compared with host linear transcripts, the circRNAs within several pathways are disproportionately upregulated in active TB patients, including "Cytokine-cytokine receptor interaction", "Chemokine signaling pathway", "Neurotrophin signaling pathway", and "Bacterial invasion of epithelial cells". Based on the differentially expressed circRNAs within these pathways, we developed a PBMC circRNA-based molecular signature differentiating active TB patients from healthy controls. We validated the classification power of the PBMC circRNA signature in an independent cohort with the area under the receiver operating characteristic curve (AUC) at 0.946. INTERPRETATION: Our results suggest that PBMC circRNAs are potentially reliable marker molecules in TB diagnosis.

TLR4-NOX2 axis regulates the phagocytosis and killing of Mycobacterium tuberculosis by macrophages.

BACKGROUND: Macrophages stand at the forefront of both innate and adapted immunity through their capacities to recognize, engulf, and eliminate foreign particles, and to stimulate adapted immune cells. They are also involved in controlling pro- and anti-inflammatory pathways. Macrophage activity against Mycobacterium tuberculosis (M. tuberculosis) has been shown to involve Toll-like receptor (TLR) activation and ROS production. Previous studies have shown that lipopolysaccharide (LPS), through TLR4, could activate macrophages, improve their bactericidal ROS production, and facilitate anti-infective immune responses. We sought to better understand the role of the TLR4-NOX2 axis in macrophage activation during M. tuberculosis infection. METHODS: THP-1 macrophages and PMA primed THP-1 macrophages [THP-1(A)] were treated with LPS and infected by M. tuberculosis. Cells were analyzed by flow cytometry for TLR4 expression, ROS production, phagocytosis, and killing of M. tuberculosis. Western blotting was used to analyze NOX2 expression. Inhibitors of the TLR4-NOX2 pathway were used to assess this pathway's role in these processes, and their role in LPS activation of macrophages. RESULTS: We found that THP1-derived macrophages or PMA primed THP-1 macrophages exhibit higher surface TLR4 levels and increased NOX2 expression levels following LPS treatment. M. tuberculosis infection reduced these levels, but LPS was able to limit the negative effects of M.tb. Additionally, LPS increases THP-1(A) cells' bactericidal activities including phagocytosis, ROS production, and destruction of M. tuberculosis. Significantly, all of these activities are impaired when TLR4 or NOX2 are inhibited. CONCLUSION: These studies demonstrate the importance of the TLR4-NOX2 axis in M. tuberculosis elimination by macrophages and may lead to novel therapies for tuberculosis and other bacterial infections.

Association between HLA-DQ Gene Polymorphisms and HBV-Related Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related mortality worldwide. Host gene variants may influence hepatitis B virus- (HBV-) related HCC. Human leukocyte antigens (HLA) play an important role in presenting virus antigens to immune cells that are responsible for the clearance of virus-infected cells and tumor cells. Previous studies have investigated the HLA-DQ (rs2856718 and rs9275572) polymorphisms that may be associated with the development of HBV-related HCC. However, the results are controversial or inconclusive. Hence, we conducted a meta-analysis to derive a more precise estimation of the associations. A total of 6 articles were used to evaluate the effect of the two polymorphisms on the risk of HBV-related HCC. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. We found that rs2856718 and rs9275572 in HLA-DQ significantly decreased HBV-related HCC in total population, especially in Chinese, but not in Saudi Arabian. Further validation of our results in larger populations and different ethnicities are required.

Expression of nuclear factor, erythroid 2-like 2-mediated genes differentiates tuberculosis.

During infection and host defense, nuclear factor, erythroid 2-like 2 (Nrf2) dependent signaling is an efficient antioxidant defensive mechanism used by host cells to control the destructive effects of reactive oxygen species. This allows for effective defense responses against microbes while minimizing oxidative injury to the host cell itself. As a central regulator of antioxidant genes, Nrf2 has gained great attention in its pivotal role in infection, especially in tuberculosis (TB), the top infectious disease killer worldwide. To elucidate the genes potentially regulated by Nrf2 in TB, we conducted a meta-analysis on published gene expression datasets. Firstly, we compared the global gene expression profiles between control and Nrf2-deficient human cells. The differentially expressed genes were deemed as "Nrf2-mediated genes". Next, the whole blood gene expression pattern of TB patients was compared with that of healthy controls, pneumonia patients, and lung cancer patients. We found that the genes deregulated in TB significantly overlap with the Nrf2-mediated genes. Based on the intersection of Nrf2-mediated and TB-regulated genes, we identified an Nrf2-mediated 17-gene signature, which reflects a cluster of gene ontology terms highly related to TB physiology. We demonstrated that the 17-gene signature can be used to distinguish TB patients from healthy controls and patients with latent TB infection, pneumonia, or lung cancer. Also, the Nrf2-mediated gene signature can be used as an indicator of the anti-TB therapeutic response. More importantly, we confirmed that the predictive power of the Nrf2-mediated 17-gene signature is significantly better than the random gene sets selected from the human transcriptome. Also, the 17-gene signature performs even better than the random gene signatures selected from TB-associated genes. Our study confirms the central role of Nrf2 in TB pathogenesis and provides a novel and useful diagnostic method to differentiate TB patients from other human subjects.

Nuclear factor, erythroid 2-like 2-associated molecular signature predicts lung cancer survival.

Nuclear factor, erythroid 2-like 2 (NFE2L2), a transcription factor also known as NF-E2-related factor 2 (Nrf2), is a key cytoprotective gene that regulates critical antioxidant and stress-responsive genes. Nrf2 has been demonstrated to be a promising therapeutic target and useful biomarker in malignant disease. We hypothesized that NFE2L2-mediated gene expression would reflect cancer severity and progression. We conducted a meta-analysis of microarray data for 240 NFE2L2-mediated genes that were enriched in tumor tissues. We then developed a risk scoring system based on NFE2L2 gene expression profiling and designated 50 tumor-associated genes as the NFE2L2-associated molecular signature (NAMS). We tested the relationship between this gene expression signature and both recurrence-free survival and overall survival in lung cancer patients. We find that NAMS predicts clinical outcome in the training cohort and in 12 out of 20 validation cohorts. Cox proportional hazard regressions indicate that NAMS is a robust prognostic gene signature, independent of other clinical and pathological factors including patient age, gender, smoking, gene alteration, MYC level, and cancer stage. NAMS is an excellent predictor of recurrence-free survival and overall survival in human lung cancer. This gene signature represents a promising prognostic biomarker in human lung cancer.

[H2O2 promotes neutrophil adherence and injury of human umbilical vein endothelial cells].

To explore the role of hydrogen peroxide (H2O2) in promoting polymorphonuclear neutrophils adherence and injury of human umbilical vein endothelial cells (HUVECs), the ordinary optical microscope and scanning electron microscopy were used to observe the adherence and injury after HUVECs co-cultured with neutrophils pretreated by extracellular H2O2 (HUVECs and neutrophils co-culture without H2O2 pretreatment as control), and the adhesion rates of neutrophils were measured through cell count test. The percentages of HUVECs expressing intercellular adhesion molecule 1 (ICAM-1) and Apo2.7 were detected by flow cytometry. After being cocultured with the neutrophils pretreated by extracellular H2O2, HUVECs showed obvious injury changes, such as round or oval shape, shortened or disappeared microvilli, and membrane structural damage; The adhesion rate of neutrophils was (57.74 ± 9.18)%, which was significantly higher than that in control [(23.12 ± 6.43)%, P < 0.01, n = 8]; The percentages of HUVECs expressing ICAM-1 and Apo2.7 were (44.69 ± 1.52)% and (39.29 ± 1.81)% respectively, which were significantly higher than those in control [(21.79 ± 1.43)% and (9.79 ± 1.43)%] (P < 0.01, n = 8). The results suggest that extracellular H2O2 can promote the neutrophils adherence and injury of HUVECs.