Comprehensive identification of immuno-related transcriptional signature for active pulmonary tuberculosis by integrated analysis of array and single cell RNA-seq.

BACKGROUND: Tuberculosis (TB) continues to be a major cause of morbidity and mortality worldwide. However, the molecular mechanism underlying immune response to human infection with Mycobacterium tuberculosis (Mtb) remains unclear. Assessing changes in transcript abundance in blood between health and disease on a genome-wide scale affords a comprehensive view of the impact of Mtb infection on the host defense and a reliable way to identify novel TB biomarkers. METHODS: We combined expression profiling by array and single cell RNA-sequencing (scRNA-seq) via 10X Genomics platform to better illustrate the immuno-related transcriptional signature of TB and explore potential diagnostic markers for differentiating TB from latent tuberculosis infection (LTBI) and healthy control (HC). FINDINGS: Pathway analysis based on differential expressed genes (DEGs) revealed that immune transcriptional profiling could effectively differ TB with LTBI and HC. Following WGCNA and PPI network analysis based on DEGs, we screened out three key immuno-related hub genes (ADM, IFIT3 and SERPING1) highly associated with TB. Further validation found only ADM expression significantly increased in TB patients in both adult and children's datasets. By comparing the scRNA-seq datasets from TB, LTBI and HC, we observed a remarkable elevated expression level and proportion of ADM in TB Myeloid cells, further supporting that ADM expression changes could distinguish patients with TB from LTBI and HC. Besides, the hsa-miR-24-3p-NEAT1-ADM-CEBPB regulation pathway might be one of the critical networks regulating the pathogenesis of TB. Although further investigation in a larger cohort is warranted, we provide useful and novel insight to explore the potential candidate genes for TB diagnosis and intervention. INTERPRETATION: We propose that the expression of ADM in peripheral blood could be used as a novel biomarker for differentiating TB with LTBI and HC.

NF-κB-mediated TET2-dependent TNF promoter demethylation drives Mtb-upregulation TNF expression in macrophages.

Tumor necrosis factor (TNF) is essential for the host defense against tuberculosis (TB). However, scarcity or excessive TNF production in macrophages can also increase susceptibility to TB. The precise mechanisms underlying how Mycobacterium tuberculosis (Mtb) induces TNF over-expression are unclear. Here, we show that Mtb infection significantly increases 5-hydroxylmethylocytosine (5hmC) levels in the TNF promoter. Luciferase reporter assays identify the precise methylated CpG sites that are essential to regulating TNF promoter activity. Infection simultaneously promotes the expression of the TET2 demethylase in macrophages. After inhibiting NF-κB or knocking down TET2, we found that TNF promoter demethylation levels is increased while Mtb-induced TNF expression decrease. Here, NF-κB binds to TET2 and mediates its recruitment to the TNF promoter to induce TNF demethylation. Finally, we show that TLR2 activation during Mtb infection promotes NF-κB translocation into the nucleus which is important for NF-κB-mediated TET2-dependent TNF promoter demethylation thus helps drive Mtb-induced TNF expression. Targeting this axis might be a novel strategy for host-directed therapy against TB.

Current approaches in laboratory testing for SARS-CoV-2.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which began in Wuhan, Hubei Province, China, has rapidly spread to produce a global pandemic. It is now clear that person-to-person transmission of SARS-CoV-2 has been occurring and that the virus has been dramatically growing in recent months. Early, rapid and accurate diagnosis is of great significance for curtailing the spread of SARS-CoV-2. There are currently several diagnostic techniques (e.g. viral culture and nucleic acid amplification test) being used to detect the virus. However, the sensitivity and specificity of these methods are quite different, with the sample source and detection limit varying greatly. This study reviewed all types and characteristics of the currently available laboratory diagnostic assays for detecting SARS-CoV-2 infection and summarized the selection strategies of testing and sampling sites at different disease stages to improve the diagnostic accuracy of Coronavirus Disease 2019 (COVID-19).

Cutting Edge: Characterization of Human Tissue-Resident Memory T Cells at Different Infection Sites in Patients with Tuberculosis.

Tissue-resident memory T cells (TRMs) have a key role in mediating the host defense against tuberculosis (TB) in mice, but their human counterparts have not been well characterized. In this article, we recruited patients with TB and determined TRM frequency, trafficking, activation marker expression, and cytokine production by flow or mass cytometry at different infection sites, including peripheral blood, pleural fluid, bronchoalveolar lavage fluid, and lung. We found a high frequency of TRMs at all infection sites apart from the peripheral blood. These TRMs exhibited a memory phenotype, were highly activated (based on CD38 and HLA-DR expression), and expressed high levels of trafficking (CCR5 and CXCR6) and exhaustion (PD-1) markers. When stimulated with Mycobacterium tuberculosis, TRMs secreted cytokines, including IFN-γ, TNF-α, and IL-2, and exhibited a multifunctional phenotype. TRMs limited intracellular M. tuberculosis replication in macrophages. These data inform our current understanding of immunosurveillance at different infection sites in patients with TB.

Ibrutinib suppresses intracellular mycobacterium tuberculosis growth by inducing macrophage autophagy.

Tuberculosis (TB) is a major cause of morbidity and mortality worldwide. The host-directed therapy is a promising strategy for TB treatment that synergize with anti-TB treatment drugs. In this study, we found that the anti-chronic lymphocytic leukemia drug, ibrutinib, inhibited the growth of intracellular Mtb in human macrophages. Mechanisms studies showed that ibrutinib treatment significantly decreased p62 and increased LC3b proteins in Mtb infected macrophages. In addition, ibrutinib increased LC3b colocalization with intracellular Mtb and auto-lysosome fusion. Furthermore, inhibition of autophagy by using siRNA targeting ATG7 abolished the effect of ibrutinib-mediated suppression of intracellular Mtb. Next, we found that ibrutinib induced autophagy was through inhibition of BTK/Akt/mTOR pathway. Finally, we confirmed that ibrutinib treatment significantly reduced Mtb load in mediastinal node and spleen of Mtb infected mice. In conclusion, our data suggest that ibrutinib is a potential host-directed therapy candidate against TB.

Single-cell transcriptomics of blood reveals a natural killer cell subset depletion in tuberculosis.

BACKGROUND: Tuberculosis (TB) continues to be a critical global health problem, which killed millions of lives each year. Certain circulating cell subsets are thought to differentially modulate the host immune response towards Mycobacterium tuberculosis (Mtb) infection, but the nature and function of these subsets is unclear. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from healthy controls (HC), latent tuberculosis infection (LTBI) and active tuberculosis (TB) and then subjected to single-cell RNA sequencing (scRNA-seq) using 10 × Genomics platform. Unsupervised clustering of the cells based on the gene expression profiles using the Seurat package and passed to tSNE for clustering visualization. Flow cytometry was used to validate the subsets identified by scRNA-Seq. FINDINGS: Cluster analysis based on differential gene expression revealed both known and novel markers for all main PBMC cell types and delineated 29 cell subsets. By comparing the scRNA-seq datasets from HC, LTBI and TB, we found that infection changes the frequency of immune-cell subsets in TB. Specifically, we observed gradual depletion of a natural killer (NK) cell subset (CD3-CD7+GZMB+) from HC, to LTBI and TB. We further verified that the depletion of CD3-CD7+GZMB+ subset in TB and found an increase in this subset frequency after anti-TB treatment. Finally, we confirmed that changes in this subset frequency can distinguish patients with TB from LTBI and HC. INTERPRETATION: We propose that the frequency of CD3-CD7+GZMB+ in peripheral blood could be used as a novel biomarker for distinguishing TB from LTBI and HC. FUND: The study was supported by Natural Science Foundation of China (81770013, 81525016, 81772145, 81871255 and 91942315), National Science and Technology Major Project (2017ZX10201301), Science and Technology Project of Shenzhen (JCYJ20170412101048337) and Guangdong Provincial Key Laboratory of Regional Immunity and Diseases (2019B030301009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Autoantibody-Mediated Erythrophagocytosis Increases Tuberculosis Susceptibility in HIV Patients.

Macrophage dysfunction is associated with increased tuberculosis (TB) susceptibility in patients with human immunodeficiency virus (HIV) infection. However, the mechanisms underlying how HIV infection impairs macrophage function are unclear. Here, we found that levels of autoantibodies against red blood cells (RBCs) were significantly elevated in patients with HIV as determined by direct antiglobulin test (DAT). DAT positivity was significantly associated with TB incidence in both univariate and multivariate analyses (odds ratio [OR] = 11.96 [confidence interval {CI}, 4.68 to 30.93] and 12.65 [3.33 to 52.75], respectively). Ex vivo analysis showed that autoantibodies against RBCs enhanced erythrophagocytosis and thus significantly impaired macrophage bactericidal function against intracellular Mycobacterium tuberculosis Mechanistically, autoantibody-mediated erythrophagocytosis increased heme oxygenase-1 (HO-1) expression, which inhibited M. tuberculosis-induced autophagy in macrophages. Silencing ATG5, a key component for autophagy, completely abrogated the effect of erythrophagocytosis on macrophage bactericidal activity against M. tuberculosis In conclusion, we have demonstrated that HIV infection increases autoantibody-mediated erythrophagocytosis. This process impairs macrophage bactericidal activity against M. tuberculosis by inhibiting HO-1-associated autophagy. These findings reveal a novel mechanism as to how HIV infection increases TB susceptibility.IMPORTANCE HIV infection significantly increases TB susceptibility due to CD4 T-cell loss and macrophage dysfunction. Although it is relatively clear that CD4 T-cell loss represents a direct effect of HIV infection, the mechanism underlying how HIV infection dampens macrophage function is unknown. Here, we show that HIV infection enhances autoantibody-mediated erythrophagocytosis, which dampens macrophage bactericidal activity against TB by inhibiting HO-1-associated autophagy. Our findings reveal a novel mechanism explaining how HIV infection increases susceptibility to TB. We propose that DAT could be a potential measure to identify HIV patients who are at high TB risk and who would be suitable for anti-TB chemotherapy preventive treatment.

Histone deacetylase inhibitors impair the host immune response against Mycobacterium tuberculosis infection.

Histone deacetylase inhibitors (HDACi), a novel class of anti-cancer drug, have been recently reported to suppress host immunity and increase susceptibility to infection. Tuberculosis, a leading infectious disease killer caused by Mycobacterium tuberculosis (M.tb), is basically the product of the interaction between bacterial virulence and host resistance. However, the effects of HDACi in host immunity against M.tb is largely unknown. In this study, we found that HDACi including Trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) significantly impaired phagocytosis and killing activity of macrophage. In line with these findings, we noted that M.tb induced reactive oxygen species (ROS) production and autophagy are significantly suppressed by TSA. Transcriptome analysis revealed that the suppression of autophagy by TSA might due to its inhibiting autophagy-regulating genes such as CACNA2D3, which regulates intracellular Ca2+ levels. Finally, we confirmed that HDACi including TSA and SAHA significantly exacerbated the histopathological damage and M.tb load in the lung of M.tb infected mice. Taken together, our results indicated that HDACi at least TSA and SAHA significantly impaired macrophage immunity against M.tb and therefore increase susceptibility to TB, our findings raised the concern that the potential side effects of HDACi on latent TB reactivation should be considered in clinic.

Regulation of Pol II Pausing Is Involved in Daily Gene Transcription in the Mouse Liver.

The circadian clock orchestrates gene expression rhythms. Regulation at the level of gene transcription is essential for molecular and cellular rhythms. Pol II pause release is a critical step of transcription regulation. However, whether and how Pol II pause release is regulated during daily transcription have not been characterized. In this study, we performed Pol II ChIP-seq across the day in the mouse liver and quantitatively analyzed binding signals within the transcription start site (TSS) region and the gene body. We frequently found discordant changes between Pol II near the TSS ([Pol II]TSS, paused Pol II) and that within the gene body ([Pol II]GB, transcribing Pol II) across the genome, with only [Pol II]GB always reflecting transcription of clock and clock-controlled genes. Accordingly, Pol II traveling ratios of more than 7000 genes showed significant daily changes (>1.5-fold). Therefore, there is widespread regulation of Pol II pausing in the mouse liver. Interestingly, gene transcription rhythms exhibited a bimodal phase distribution. The transcription of ~400 genes peaked near ZT0, coincident with a genome-wide increase in [Pol II]TSS and traveling ratio (TR). The transcription of ~300 other genes peaked ~12 h later, when there was a global decrease in [Pol II]TSS and TR. ChIP-seq against TATA-binding protein (Tbp), a preinitiation complex (PIC) component, revealed that Pol II recruitment mainly played an indirect role in transcriptional output, with transcriptional termination and pause release functioning prominently in determining the fate of initiated Pol II and its pausing status. Taken together, our results revealed a critical, albeit complex role of Pol II pausing control in regulating the temporal output of gene transcription.

Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells.

Reactive oxygen species formed within the mammalian cell can produce 8-oxo-7,8-dihydroguanine (8-oxoG) in mRNA, which can cause base mispairing during gene expression. Here we found that administration of 8-oxoGTP in MTH1-knockdown cells results in increased 8-oxoG content in mRNA. Under this condition, an amber mutation of the reporter luciferase is suppressed. Using second-generation sequencing techniques, we found that U-to-G changes at preassigned sites of the luciferase transcript increased when 8-oxoGTP was supplied. In addition, an increased level of 8-oxoG content in RNA induced the accumulation of aggregable amyloid ß peptides in cells expressing amyloid precursor protein. Our findings indicate that 8-oxoG accumulation in mRNA can alter protein synthesis in mammalian cells. Further work is required to assess the significance of these findings under normal physiological conditions.

RNA over-editing of BLCAP contributes to hepatocarcinogenesis identified by whole-genome and transcriptome sequencing.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, although the treatment of this disease has changed little in recent decades because most of the genetic events that initiate this disease remain unknown. To better understand HCC pathogenesis at the molecular level and to uncover novel tumor-initiating events, we integrated RNA-seq and DNA-seq data derived from two pairs of HCC tissues. We found that BLCAP is novel editing gene in HCC and has over-editing expression in 40.1% HCCs compared to adjacent liver tissues. We then used RNA interference and gene transfection to assess the roles of BLCAP RNA editing in tumor proliferation. Our results showed that compared to the wild-type BLCAP gene, the RNA-edited BLCAP gene may stably promote cell proliferation (including cell growth, colony formation in vitro, and tumorigenicity in vivo) by enhancing the phosphorylation of AKT, mTOR, and MDM2 and inhibiting the phosphorylation of TP53. Our current results suggest that the RNA over-editing of BLCAP gene may serve as a novel potential driver in advanced HCC through activating AKT/mTOR signal pathway.

Global gene expression profiling identifies ALDH2, CCNE1 and SMAD3 as potential prognostic markers in upper tract urothelial carcinoma.

BACKGROUND: Current knowledge about the molecular properties and prognostic markers of upper tract urothelial carcinoma (UTUC) is sparse and often based on bladder urothelial carcinoma (UC), which is thought to share common risk factors with UTUC. However, studies have suggested that differences exist regarding tumor behavior and molecular biology of these cancers, comprehensive investigations are needed to guide the clinical management of UTUC. In recent years, massively parallel sequencing has allowed insights into the biology of many cancers, and molecular prognostic markers based on this approach are rapidly emerging. The goal of this study was to characterize the gene expression patterns of UTUC using massively parallel sequencing, and identify potential molecular markers for prognosis in patients with UTUC. METHODS: We compared the genome-wide mRNA expression profile of cancer and matched normal tissues from 10 patients with UTUC to identify significantly deregulated genes. We also examined the protein levels of prognostic marker candidates in 103 patients with UTUC, and tested the association of these markers with overall survival using Kaplan-Meier model and Cox regression. RESULTS: Functional enrichment of significantly deregulated genes revealed that expression patterns of UTUC were characterized by disorders of cell proliferation and metabolism. And we also compared the expression profile of UTUC with that of bladder UC. Our results highlighted both shared (e.g. disorders of cell cycling and growth signal transduction) and tumor-specific (e.g. abnormal metabolism in UTUC and disruptions of adhesion pathways in bladder UC) features of these two cancers. Importantly, we identified that low protein expression of ALDH2 while high CCNE1 and SMAD3 were significantly associated with increased depth (*P <0.05) and lower overall survival (***P <0.0001) in an independent set of 103 patients. Multivariate Cox regression revealed that all these three genes were independent prognostic indicators in patients with UTUC (***P <0.001). CONCLUSIONS: In conclusion, our study characterized the comprehensive expression profile of UTUC and highlighted both commons and differences in expression patterns between UTUC and bladder UC. And we, for the first time, revealed that ALDH2, CCNE1 and SMAD3 are associated with prognosis in patients with UTUC.

Multilayered molecular profiling supported the monoclonal origin of metastatic renal cell carcinoma.

Primary renal cell carcinomas (pRCCs) have a high degree of intratumoral heterogeneity and are composed of multiple distinct subclones. However, it remains largely unknown that whether metastatic renal cell carcinomas (mRCCs) also have startling intratumoral heterogeneity or whether development of mRCCs is due to early dissemination or late diagnosis. To decipher the evolution of mRCC, we analyzed the multilayered molecular profiles of pRCC, local invasion of the vena cava (IVC), and distant metastasis to the brain (MB) from the same patient using whole-genome sequencing, whole-exome sequencing, DNA methylome profiling, and transcriptome sequencing. We found that mRCC had a lower degree of heterogeneity than pRCC and was likely to result from recent clonal expansion of a rare, advantageous subclone. Consequently, some key pathways that are targeted by clinically available drugs showed distinct expression patterns between pRCC and mRCC. From the genetic distances between different tumor subclones, we estimated that the progeny subclone giving rise to distant metastasis took over half a decade to acquire the full potential of metastasis since the birth of the subclone that evolved into IVC. Our evidence supported that mRCC was monoclonal and distant metastasis occurred late during renal cancer progression. Thus, there was a broad window for early detection of circulating tumor cells and future targeted treatments for patients with mRCCs should rely on the molecular profiles of metastases.

A systematic analysis on DNA methylation and the expression of both mRNA and microRNA in bladder cancer.

BACKGROUND: DNA methylation aberration and microRNA (miRNA) deregulation have been observed in many types of cancers. A systematic study of methylome and transcriptome in bladder urothelial carcinoma has never been reported. METHODOLOGY/PRINCIPAL FINDINGS: The DNA methylation was profiled by modified methylation-specific digital karyotyping (MMSDK) and the expression of mRNAs and miRNAs was analyzed by digital gene expression (DGE) sequencing in tumors and matched normal adjacent tissues obtained from 9 bladder urothelial carcinoma patients. We found that a set of significantly enriched pathways disrupted in bladder urothelial carcinoma primarily related to "neurogenesis" and "cell differentiation" by integrated analysis of -omics data. Furthermore, we identified an intriguing collection of cancer-related genes that were deregulated at the levels of DNA methylation and mRNA expression, and we validated several of these genes (HIC1, SLIT2, RASAL1, and KRT17) by Bisulfite Sequencing PCR and Reverse Transcription qPCR in a panel of 33 bladder cancer samples. CONCLUSIONS/SIGNIFICANCE: We characterized the profiles between methylome and transcriptome in bladder urothelial carcinoma, identified a set of significantly enriched key pathways, and screened four aberrantly methylated and expressed genes. Conclusively, our findings shed light on a new avenue for basic bladder cancer research.

Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma.

We sequenced whole exomes of ten clear cell renal cell carcinomas (ccRCCs) and performed a screen of ∼1,100 genes in 88 additional ccRCCs, from which we discovered 12 previously unidentified genes mutated at elevated frequencies in ccRCC. Notably, we detected frequent mutations in the ubiquitin-mediated proteolysis pathway (UMPP), and alterations in the UMPP were significantly associated with overexpression of HIF1α and HIF2α in the tumors (P = 0.01 and 0.04, respectively). Our findings highlight the potential contribution of UMPP to ccRCC tumorigenesis through the activation of the hypoxia regulatory network.