Single-cell transcriptomics of blood identified IFIT1+ neutrophil subcluster expansion in NTM-PD patients.

OBJECTIVE: Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is caused by an imbalance between pathogens and impaired host immune responses. Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) are the two major pathogens that cause NTM-PD. In this study, we sought to dissect the transcriptomes of peripheral blood immune cells at the single-cell resolution in NTM-PD patients and explore potential clinical markers for NTM-PD diagnosis and treatment. METHODS: Peripheral blood samples were collected from six NTM-PD patients, including three MAB-PD patients, three MAC-PD patients, and two healthy controls. We employed single-cell RNA sequencing (scRNA-seq) to define the transcriptomic landscape at a single-cell resolution. A comprehensive scRNA-seq analysis was performed, and flow cytometry was conducted to validate the results of scRNA-seq. RESULTS: A total of 27,898 cells were analyzed. Nine T-cells, six mononuclear phagocytes (MPs), and four neutrophil subclusters were defined. During NTM infection, naïve T-cells were reduced, and effector T-cells increased. High cytotoxic activities were shown in T-cells of NTM-PD patients. The proportion of inflammatory and activated MPs subclusters was enriched in NTM-PD patients. Among neutrophil subclusters, an IFIT1+ neutrophil subcluster was expanded in NTM-PD compared to healthy controls. This suggests that IFIT1+ neutrophil subcluster might play an important role in host defense against NTM. Functional enrichment analysis of this subcluster suggested that it is related to interferon response. Cell-cell interaction analysis revealed enhanced CXCL8-CXCR1/2 interactions between the IFIT1+ neutrophil subcluster and NK cells, NKT cells, classical mononuclear phagocytes subcluster 1 (classical Mo1), classical mononuclear phagocytes subcluster 2 (classical Mo2) in NTM-PD patients compared to healthy controls. CONCLUSIONS: Our data revealed disease-specific immune cell subclusters and provided potential new targets of NTM-PD. Specific expansion of IFIT1+ neutrophil subclusters and the CXCL8-CXCR1/2 axis may be involved in the pathogenesis of NTM-PD. These insights may have implications for the diagnosis and treatment of NTM-PD.

Armcx1 Reduces Neurological Damage Via a Mitochondrial Transport Pathway Involving Miro1 After Traumatic Brain Injury.

Armcx1 is a member of the ARMadillo repeat-Containing protein on the X chromosome (ARMCX) family, which is recognized to have evolutionary conserved roles in regulating mitochondrial transport and dynamics. Previous research has shown that Armcx1 is expressed at higher levels in mice after axotomy and in adult retinal ganglion cells after crush injury, and this protein increases neuronal survival and axonal regeneration. However, its role in traumatic brain injury (TBI) is unclear. Therefore, the aim of this study was to assess the expression of Armcx1 after TBI and to explore possible related mechanisms by which Armcx1 is involved in TBI. We used C57BL/6 male mice to model TBI and evaluated the role of Armcx1 in TBI by transfecting mice with Armcx1 small interfering RNA (siRNA) to inhibit Armcx1 expression 24 h before TBI modeling. Western blotting, immunofluorescence, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, Nissl staining, transmission electron microscopy, adenosine triphosphate (ATP) level measurement, neuronal apoptosis analysis, neurological function scoring and the Morris water maze were performed. The results demonstrated that Armcx1 protein expression was elevated after TBI and that the Armcx1 protein was localized in neurons and astroglial cells in cortical tissue surrounding the injury site. In addition, inhibition of Armcx1 expression further led to impaired mitochondrial transport, abnormal morphology, reduced ATP levels, aggravation of neuronal apoptosis and neurological dysfunction, and decrease Miro1 expression. In conclusion, our findings indicate that Armcx1 may exert neuroprotective effects by ameliorating neurological injury after TBI through a mitochondrial transport pathway involving Miro1.

First report of anthracnose caused by Colletotrichum kahawae on Hypericum chinensis in China.

Hypericum chinensis is growing in popularity amongst consumers in cut-flower and pop-flower market as an ornamental woody plant for its florid berry and colorful flower. In August 2019, a new leaf spot disease was observed on H. chinensis in three commercial nurseries in Kunming (25°05'N, 102°72'E), Yunnian province, China. Disease symptoms were observed on approximately 40% of the plants one year after planting and 30% of the leaves were infected. Leaf symptoms began as small, water-soaked lesions on young leaves which later became larger, dark brown and necrotic. The lesion size ranged from 0.2 to 2.8 cm in diameter. For pathogen isolation, three samples of symptomatic leaves were collected from four different nurseries. The leaves were cut into 0.5 mm pieces, surface sterilized using 70% ethanol for 30 s, and 3% NaOCl for 5 min, rinsed three times in sterilized distilled water and plated on potato dextrose agar (PDA) (Zhou et al. 2023). The plates were incubated at 26°C in the dark for 3 days. Eight isolates with comparable morphological characteristics were obtained. Initially, colonies produced pale gray to white aerial mycelia, turning dark gray after 5 days. The isolates produced hyaline, single celled, straight and cylindrical conidia, with mean size 9.7 to 14.8 µm long × 3.7 to 5.6 µm wide (n = 100). Morphological characteristics were consistent with Colletotrichum sp. (Bailey and Jeger 1992). For molecular analysis, genomic DNA was extracted from three representative isolates (XSD1, XSD3 and XSD5), amplified using the primers ITS1/ITS4 (Yin et al. 2012) and T1/Bt2b (Glass and Donaldson 1995) and submitted to sequencing (Weir et al. 2012). DNA sequences of the isolates XSD2, XSD3 and XSD8 were identical. DNA sequences of a representative isolate XSD2 were deposited in GenBank (accession no. MW202334 for ITS, and OR347007 for TUB 2). MegaBLAST analysis of the ITS and TUB2 sequences showed 99.5% and 99.3% similarity with C. kahawae strain ICMP 18539 (accession no. NR_120138.1 for ITS) and strain IMI319418 (JX145227.1 for TUB 2). Pathogenicity tests were conducted by inoculating the pathogen on healthy mature leaves of H. chinensis in the field. Ten leaves (two leaves/plant) were inoculated by spraying conidial suspension (106 spores/ml) of isolates XSD1, XSD3 and XSD5, and covered with plastic bags to maintain high humidity for 48 hours, respectively. Leaves treated with sterile distilled water served as a control. All inoculated leaves showed symptoms similar to those observed in the field at 23±5°C 10 days after inoculation. No symptoms developed on non-inoculated leaves. The pathogen was re-isolated from inoculated diseased leaves and identified as C. kahawae based on morphological and molecular characters. C. kahawae has been reported to cause leaf spot on cultivated rocket in Italy (Garibaldi et al. 2016), and anthracnose disease on tree tomato in Colombia (Rojas et al. 2018), to our knowledge, this is the first report of C. kahawae causing anthracnose on H. chinensis worldwide. Due to important ornamental and economic value of H. chinensis, the distribution of C. kahawae needs to be investigated and monitored for effective disease management strategies to be developed.

Macrophage targeted iron oxide nanodecoys augment innate immunological and drug killings for more effective Mycobacterium Tuberculosis clearance.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, is still one of the top killers worldwide among infectious diseases. The escape of Mtb from immunological clearance and the low targeting effects of anti-TB drugs remain the substantial challenges for TB control. Iron is particularly required for Mtb growth but also toxic for Mtb in high dosages, which makes iron an ideal toxic decoy for the 'iron-tropic' Mtb. Here, a macrophage-targeted iron oxide nanoparticles (IONPs)-derived IONPs-PAA-PEG-MAN nanodecoy is designed to augment innate immunological and drug killings against intracellular Mtb. IONPs-PAA-PEG-MAN nanodecoy exhibits preferential uptake in macrophages to significantly increase drug uptake with sustained high drug contents in host cells. Moreover, it can serve as a specific nanodecoy for the 'iron-tropic' Mtb to realize the localization of Mtb contained phagosomes surrounding the drug encapsulated nanodecoys and co-localization of Mtb with the drug encapsulated nanodecoys in lysosomes, where the incorporated rifampicin (Rif) can be readily released under acidic lysosomal condition for enhanced Mtb killing. This drug encapsulated nanodecoy can also polarize Mtb infected macrophages into anti-mycobacterial M1 phenotype and enhance M1 macrophage associated pro-inflammatory cytokine (TNF-α) production to trigger innate immunological responses against Mtb. Collectively, Rif@IONPs-PAA-PEG-MAN nanodecoy can synergistically enhance the killing efficiency of intracellular Mtb in in vitro macrophages and ex vivo monocyte-derived macrophages, and also significantly reduce the mycobacterial burdens in the lung of infected mice with alleviated pathology. These results indicate that Rif@IONPs-PAA-PEG-MAN nanodecoy may have a potential for the development of more effective therapeutic strategy against TB by manipulating augmented innate immunity and drug killings.

Mycobacterium tuberculosis infection depressed cytotoxic T cells activity owing to decreasing NKG2C and increasing NKG2A expression.

Cytotoxic T lymphocytes (CTLs) play protective roles in immunity against tuberculosis (TB) infection by strongly inhibiting intracellular mycobacterial growth. In TB infection, the impairing mechanism of CTLs function remains unclear. In this study, we identified that the cytotoxic granule molecules expression levels of perforin (PRF) and granulysin (GNLY) in CD3+ and CD8+ CTL cells were significantly depressed in TB patients compared to those in healthy donors. The frequencies of T-CTLs, co-expressing granzyme B (GZMB), PRF and GNLY, were obviously decreased in TB patients. Moreover, NKG2C highly expressed in T-CTLs, was an effective activator of cytotoxic activity of CD3+ T cells. And, NKG2C+CD3+ T cells potently inhibited intracellular mycobacterial growth. The proportions of NKG2C+ cells in CD3+ and CD8+ T cells were dramatically decreased in TB patients. Contrarily, NKG2A, an inhibitor of T cells cytotoxic activities, was highly expressed in T-CTLs of CD3+ and CD8+ T cells in TB patients. Here, we successfully discovered that depressed CTLs activities in TB patients were attributed to low expression of cytotoxic granule molecules and high expression of inhibitory NKG2A receptor, suppression of agonist receptor NKG2C. Thus, NKG2 receptors were potential targets for immunotherapy of tuberculosis, especially for multidrug-resistant tuberculosis.

Circular RNA circTRAPPC6B Enhances IL-6 and IL-1ß Expression and Repolarizes Mycobacteria Induced Macrophages from M2- to M1-Like Phenotype by Targeting miR-892c-3p.

Mycobacterium tuberculosis (Mtb) infection elicits macrophage polarization into M2 phenotype to block the host's protective immune response. However, it remains unclear how Mtb regulates macrophage polarization. Recent studies have suggested that noncoding RNA may play a role in macrophage polarization. In this study, we investigated the potential involvement of circTRAPPC6B, a circular RNA that is downregulated in tuberculosis (TB) patients, in regulating macrophage polarization. We found that Mtb infection downregulated M1-related IL-6 and IL-1ß while highly expressed M2-related CCL22 and CD163. Overexpressed circTRAPPC6B had switched Mtb-infected macrophages from M2- to M1-like phenotype, accompanied by upregulation of IL-6 and IL-1ß. Meanwhile overexpressed circTRAPPC6B significantly inhibited Mtb growth in macrophages. Our findings suggest that circTRAPPC6B may regulate macrophage polarization by targeting miR-892c-3p, which is highly expressed in TB patients and M2-like macrophages. And miR-892c-3p inhibitor decreased intracellular Mtb growth in macrophages. Thus, TB-inhibited circTRAPPC6B could specifically induce IL-6 and IL-1ß expression to switch/antagonize Mtb-induced macrophage polarization from M2- to M1-like phenotype by targeting miR-892c-3p, leading to enhanced host clearance of Mtb. Our results reveal a potential role for circTRAPPC6B in regulating macrophage polarization during Mtb infection and provide new insights into the molecular mechanisms underlying host defense against Mtb.

Dietary palmitoleic acid reprograms gut microbiota and improves biological therapy against colitis.

Magnitude and diversity of gut microbiota and metabolic systems are critical in shaping human health and diseases, but it remains largely unclear how complex metabolites may selectively regulate gut microbiota and determine health and diseases. Here, we show that failures or compromised effects of anti-TNF-α therapy in inflammatory bowel diseases (IBD) patients were correlated with intestinal dysbacteriosis with more pro-inflammatory bacteria, extensive unresolved inflammation, failed mucosal repairment, and aberrant lipid metabolism, particularly lower levels of palmitoleic acid (POA). Dietary POA repaired gut mucosal barriers, reduced inflammatory cell infiltrations and expressions of TNF-α and IL-6, and improved efficacy of anti-TNF-α therapy in both acute and chronic IBD mouse models. Ex vivo treatment with POA in cultured inflamed colon tissues derived from Crohn's disease (CD) patients reduced pro-inflammatory signaling/cytokines and conferred appreciable tissue repairment. Mechanistically, POA significantly upregulated the transcriptional signatures of cell division and biosynthetic process of Akkermansia muciniphila, selectively increased the growth and abundance of Akkermansia muciniphila in gut microbiota, and further reprogrammed the composition and structures of gut microbiota. Oral transfer of such POA-reprogrammed, but not control, gut microbiota induced better protection against colitis in anti-TNF-α mAb-treated recipient mice, and co-administration of POA with Akkermansia muciniphila showed significant synergistic protections against colitis in mice. Collectively, this work not only reveals the critical importance of POA as a polyfunctional molecular force to shape the magnitude and diversity of gut microbiota and therefore promote the intestinal homeostasis, but also implicates a new potential therapeutic strategy against intestinal or abenteric inflammatory diseases.

Structural insights of the elongation factor EF-Tu complexes in protein translation of Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second-deadliest infectious disease worldwide. Emerging evidence shows that the elongation factor EF-Tu could be an excellent target for treating Mtb infection. Here, we report the crystal structures of Mtb EF-Tu•EF-Ts and EF-Tu•GDP complexes, showing the molecular basis of EF-Tu's representative recycling and inactive forms in protein translation. Mtb EF-Tu binds with EF-Ts at a 1:1 ratio in solution and crystal packing. Mutation and SAXS analysis show that EF-Ts residues Arg13, Asn82, and His149 are indispensable for the EF-Tu/EF-Ts complex formation. The GDP binding pocket of EF-Tu dramatically changes conformations upon binding with EF-Ts, sharing a similar GDP-exchange mechanism in E. coli and T. ther. Also, the FDA-approved drug Osimertinib inhibits the growth of M. smegmatis, H37Ra, and M. bovis BCG strains by directly binding with EF-Tu. Thus, our work reveals the structural basis of Mtb EF-Tu in polypeptide synthesis and may provide a promising candidate for TB treatment.

Research on Herbal Therapies for Osteoarthritis in 2004-2022: A Web of Science-Based Cross-Sectional Bibliometric Analysis.

Objective: The extent, range, and nature of available research in the field of herbal therapies for osteoarthritis (OA) have not been systematically analyzed. This study aimed to map the literature available on herbal therapies for OA and identify global hotspots and trends in this field. Methods: Studies on herbal therapies for OA published between 2004 and 2022 were searched from the Web of Science Core Collection. Microsoft Excel, SPSS Statistics, and CiteSpace software were used to analyze and visualize the quantity and citations of publications, and the research hotspots and trends in research on herbal therapies for OA. Results: A total of 1649 publications mainly from 76 countries/regions and 270 institutions were included in this study. From 2004 to 2022, there is an upward trend in the publications of herbal therapies for OA. China ranked first in the number of publications (n = 568, 34.45%), followed by the USA (n = 353, 21.41%), South Korea (n = 187, 11.34%), Germany (n = 85, 5.15%), and England (n = 79, 4.79%). Kyung Hee University (n = 46), Xianxiang Liu (n = 25), and Evidence-Based Complementary and Alternative Medicine (n = 74) were the most prolific affiliation, author, and journal, respectively. Felson DT (n = 185) and Arthritis and Rheumatism (n = 1173) held the record for the most cited papers by an author and journal, respectively. Currently, the hot keywords in the field of herbal therapies for OA include knee OA, traditional Chinese medicine (TCM), differentiation, rosa canina, inflammation, oxidative stress, stem cell, and regenerative medicine. The emerging research trends in herbal therapies for OA are herbal medicinal product, chronic knee pain, mesenchymal stem cell, and clinical pharmacology. Conclusions: Research on herbal therapies for OA is flourishing, but communication among countries/regions should be strengthened. Current research on herbal therapies for OA mainly focuses on knee OA, TCM, differentiation, rosa canina, inflammation, oxidative stress, stem cell, and regenerative medicine. The research frontiers are herbal medicinal product, chronic knee pain, mesenchymal stem cell, and clinical pharmacology.

Adjunctive Zoledronate + IL-2 administrations enhance anti-tuberculosis Vγ2Vδ2 T-effector populations, and improve treatment outcome of multidrug-resistant tuberculosis1.

Multidrug-resistant tuberculosis (MDR-TB) is a refractory disease with high mortality rate due to no or few choices of antibiotics. Adjunctive immunotherapy may help improve treatment outcome of MDR-TB. Our decade-long studies demonstrated that phosphoantigen-specific Vγ2Vδ2 T cells play protective roles in immunity against TB. Here, we hypothesized that enhancing protective Vγ2Vδ2 T-effector cells could improve treatment outcome of MDR-TB. To address this, we employed clinically approved drugs Zoledronate (ZOL) and IL-2 to induce anti-TB Vγ2Vδ2 T-effector cells as adjunctive immunotherapy against MDR-TB infection of macaques. We found that adjunctive ZOL/IL-2 administrations during TB drugs treatment of MDR-TB-infected macaques significantly expanded Vγ2Vδ2 T cells and enhanced/sustained Vγ2Vδ2 T-effector subpopulation producing anti-TB cytokines until week 21. ZOL/IL-2 administrations, while expanding Vγ2Vδ2 T cells, significantly increased/sustained numbers of circulating CD4+ Th1 and CD8+ Th1-like effector populations, with some γδ T- or αß T-effector populations trafficking to airway at week 3 until week 19 or 21 after MDR-TB infection. Adjunctive ZOL/IL-2 administrations after MDR-TB infection led to lower bacterial burdens in lungs than TB drugs alone, IL-2 alone or saline controls, and resulted in milder MDR-TB pathology/lesions. Thus, adjunctive Zoledronate + IL-2 administrations can enhance anti-TB Vγ2Vδ2 T- and αß T-effector populations, and improve treatment outcome of MDR-TB.

Occurrence of Geranium wilfordii anthracnose caused by Colletotrichum dematium in China.

Geranium wilfordii Maxim. is a weed of perennial herbs and considerable medicinal plant for treating acute and chronic rheumatalgia in China. In August 2019, leaf spots on G. wilfordii were observed in Harbin (45°60'N, 126°64'E), Heilongjiang Province, China. The disease occurred on 15 to 30% of G. wilfordii leaves in three nurseries (~1.5 ha/each nursery). Initial symptoms were brown necrotic spots with a gray-white center, which enlarged gradually from approximately 1 to 5 mm in diameter, and produced concentric rings and became necrotic. Twelve infected tissues from twelve diseased leaves were surface disinfested in 0.5% NaOCl for 5 min, rinsed three times in sterile distilled water, dried on sterilized filter paper and cultured on potato dextrose agar (PDA) amended with 50 µg/ml streptomycin at 26°C for 5 days. Eight fungal cultures with consistent characteristics were obtained and subcultured by transferring hyphal tips onto fresh PDA. Single-conidium isolates were generated with methods reported previously (Leslie and Summerell 2006). Colonies on PDA consisted of cottony, dense, grayish white mycelium, pale gray colony. Conidia of a representative isolate LGC2 were single-celled, hyaline, cylindrical to slightly curved with a rounded apex and truncated base that measured 16.2 to 22.5 µm (length) × 2.6 to 3.7 µm (width) (n = 50). The appressoria were elliptic to claviform or slightly lobed on synthetic nutrient-poor agar. Based on these characteristics, the eight isolates were identified as Colletotrichum dematium (Damm et al. 2009). Genomic DNA was extracted from representative isolates LGC2, LGC3, LGC5 and the internal transcribed spacer regions (ITS)，beta-tubulin (TUB2) and actin (ACT) were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012), T1/Bt2b (Glass and Donaldson 1995) and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. DNA sequences of isolates LGC2, LGC3, and LGC5 were identical and deposited onto the GenBank (accession nos. MW193053.1 for ITS, MZ357349.1 for TUB2, and OL956946.1 for ACT). MegaBLAST analysis showed 100%, 99.7% and 100% identical to C. dematium isolates CBS 125.25 (accession nos. NR_111453.1 for ITS 552/553 bp, GU228113.1 for TUB2 386/387 bp, and GU227917.1 for ACT 231/231 bp respectively. A pathogenicity test was performed on with a representative isolate LGC2 by spraying spore suspension (1 × 106 conidia/ml) on the surfaces of all leaves of ten healthy three-month-old G. wilfordii plants. All leaves of ten control plants were inoculated with sterile water to serve as the control. All plants were placed in a humidity chamber (>95% RH, 26℃) for 48 h after inoculation and then transfered in a greenhouse at 22/28°C with a 12:12h light-dark cycle for 10 days. All inoculated leaves showed symptoms similar to those observed in the fields, while no symptoms were observed on the control leaves. The experiment was conducted twice. The fungus was re-isolated from the infected leaves and confirmed to be C. dematium according to morphological and molecular characteristics. C. dematium has previously been reported on common knotgrass (Liu et al. 2016), on piper betle (Sun et al. 2020), peanut anthracnose in China (Yu et al. 2020). To our knowledge, this is the first report of C. dematium causing G. wilfordii anthracnose in China. G. wilfordii anthracnose caused by C. dematium poses a threat to significantly reduce the quality of G. wilfordii. Therefore, its distribution needs to be investigated and effective disease management strategies developed.

Use of Whole-Genome Sequencing to Predict Mycobacterium tuberculosis Complex Drug Resistance from Early Positive Liquid Cultures.

Our objective was to evaluate the performance of whole-genome sequencing (WGS) from early positive liquid cultures for predicting Mycobacterium tuberculosis complex (MTBC) drug resistance. Clinical isolates were obtained from tuberculosis patients at Shanghai Pulmonary Hospital (SPH). Antimicrobial susceptibility testing (AST) was performed, and WGS from early Bactec mycobacterial growth indicator tube (MGIT) 960-positive liquid cultures was performed to predict the drug resistance using the TB-Profiler informatics platform. A total of 182 clinical isolates were enrolled in this study. Using phenotypic AST as the gold standard, the overall sensitivity and specificity for WGS were, respectively, 97.1% (89.8 to 99.6%) and 90.4% (83.4 to 95.1%) for rifampin, 91.0% (82.4 to 96.3%) and 95.2% (89.1 to 98.4%) for isoniazid, 100.0% (89.4 to 100.0%) and 87.3% (80.8 to 92.1%) for ethambutol, 96.6% (88.3 to 99.6%) and 61.8% (52.6 to 70.4%) for streptomycin, 86.8% (71.9 to 95.6%) and 95.8% (91.2 to 98.5%) for moxifloxacin, 86.5% (71.2 to 91.5%) and 95.2% (90.3 to 98.0%) for ofloxacin, 100.0% (54.1 to 100.0%) and 67.6% (60.2 to 74.5%) for amikacin, 100.0% (63.1 to 100.0%) and 67.2% (59.7 to 74.2%) for kanamycin, 62.5% (24.5 to 91.5%) and 88.5% (82.8 to 92.8%) for ethionamide, 33.3% (4.3 to 77.7%) and 98.3% (95.1 to 99.7%) for para-aminosalicylic acid, and 0.0% (0.0 to 12.3%) and 100.0% (97.6 to 100.0%) for cycloserine. The concordances of WGS-based AST and phenotypic AST were as follows: rifampin (92.9%), isoniazid (93.4%), ethambutol (89.6%), streptomycin (73.1%), moxifloxacin (94.0%), ofloxacin (93.4%), amikacin (68.7%), kanamycin (68.7%), ethionamide (87.4%), para-aminosalicylic acid (96.2%) and cycloserine (84.6%). We conclude that WGS could be a promising approach to predict MTBC resistance from early positive liquid cultures. IMPORTANCE In this study, we used whole-genome sequencing (WGS) from early positive liquid (MGIT) cultures instead of solid cultures to predict drug resistance of 182 Mycobacterium tuberculosis complex (MTBC) clinical isolates to predict drug resistance using the TB-Profiler informatics platform. Our study indicates that WGS may be a promising method for predicting MTBC resistance using early positive liquid cultures.

The gut microbiota mediates protective immunity against tuberculosis via modulation of lncRNA.

The gut-lung axis has been implicated as a potential therapeutic target in lung disorders. While increasing evidence suggests that gut microbiota plays a critical role in regulating host immunity and contributing to tuberculosis (TB) development and progression, the underlying mechanisms whereby gut microbiota may impact TB outcomes are not fully understood. Here, we found that broad-spectrum antibiotics treatment increased susceptibility to Mycobacterium tuberculosis (M. tuberculosis) infection and modulated pulmonary inflammatory responses in mouse M. tuberculosis infection model. We then identified a commensal gut bacteria-regulated lncRNA, termed lncRNA-CGB, which was down-regulated by dysbiosis of gut microbiota during TB infection. Furthermore, we found that Bacteroides fragilis (B. fragilis) was a direct regulator of lncRNA-CGB, and oral administration of B. fragilis enhanced expression of lncRNA-CGB and promoted anti-TB immunity. Genomic knock-out of lncRNA-CGB led to reduced IFN-γ expression and impaired anti-TB immunity, therefore leading to detrimental effects on M. tuberculosis infection. Mechanistically, lncRNA-CGB interacted with EZH2 and negatively regulated H3K27 tri-methylation (H3K27Me3) epigenetic programming, leading to enhanced IFN-γ expression. Thus, this work not only uncovered previously unrecognized importance of gut bacteria-lncRNA-EZH2-H3K27Me3 axis in conferring immune protection against TB but also identified a potential new paradigm to develop a microbiota-based treatment against TB and potentially other diseases.

Nanocages engineered from Bacillus Calmette-Guerin facilitate protective Vγ2Vδ2 T cell immunity against Mycobacterium tuberculosis infection.

Tuberculosis (TB), induced by Mycobacterium tuberculosis (Mtb) infection, remains a top killer among infectious diseases. While Bacillus Calmette-Guerin (BCG) is the sole TB vaccine, the clumped-clustered features of BCG in intradermal immunization appear to limit both the BCG protection efficacy and the BCG vaccination safety. We hypothesize that engineering of clumped-clustered BCG into nanoscale particles would improve safety and also facilitate the antigen-presenting-cell (APC)'s uptake and the following processing/presentation for better anti-TB protective immunity. Here, we engineered BCG protoplasts into nanoscale membraned BCG particles, termed as "BCG-Nanocage" to enhance the anti-TB vaccination efficiency and safety. BCG-Nanocage could readily be ingested/taken by APC macrophages selectively; BCG-Nanocage-ingested macrophages exhibited better viability and developed similar antimicrobial responses with BCG-infected macrophages. BCG-Nanocage, like live BCG bacilli, exhibited the robust capability to activate and expand innate-like T effector cell populations of Vγ2+ T, CD4+ T and CD8+ T cells of rhesus macaques in the ex vivo PBMC culture. BCG-Nanocage immunization of rhesus macaques elicited similar or stronger memory-like immune responses of Vγ2Vδ2 T cells, as well as Vγ2Vδ2 T and CD4+/CD8+ T effectors compared to live BCG vaccination. BCG-Nanocage- immunized macaques developed rapidly-sustained pulmonary responses of Vγ2Vδ2 T cells upon Mtb challenge. Furthermore, BCG- and BCG-Nanocage- immunized macaques, but not saline controls, exhibited undetectable Mtb infection loads or TB lesions in the Mtb-challenged lung lobe and hilar lymph node at endpoint after challenge. Thus, the current study well justifies a large pre-clinical investigation to assess BCG-Nanocage for safe and efficacious anti-TB vaccination, which is expected to further develop novel vaccines or adjuvants.

MIR337-3p Enhances Mycobacterial Pathogenicity Involving TLR4/MYD88 and STAT3 Signals, Impairing VDR Antimicrobial Response and Fast-Acting Immunity.

Active form of vitamin D (VitD) enhances human innate immunity against Mycobacterium tuberculosis (Mtb) infection. Our previous studies showed that MIR337-3p was highly expressed in lymphocytes of tuberculosis (TB) patients. Here, we identified the mechanism of MIR337-3p in the regulation of fast-acting anti-TB immunity by inhibiting VitD-dependent antimicrobial response pathways. While high-level MIR337-3p expression was induced by mycobacterial infection in cellular models and mice, TB patients exhibited significantly increased MIR337-3p in CD14+ monocytes/macrophages, innate-like Vγ2+ T cells, and CD8+ lymphocytes containing natural killer (NK)/innate lymphoid cells. MIR337-3p promoted the mycobacterial entry/infection and replication/growth in host target cells: macrophages and lung epithelial cells. Such MIR337-3p-enhanced pathogenicity coincided with the MIR337-3p depression of VitD-dependent antimicrobial response of cytochrome P450, family 27, subfamily b, polypeptide 1 (CYP27B1)/Beta-defensin 4 (DEFB4A)/ cathelicidin antimicrobial peptide CAMP pathways. Surprisingly, single MIR337-3p species could specifically target both the Toll-like receptor 4 (TLR4) and signal transducer and activator of transcription 3 (STAT3) 3'-untranslated regions (UTRs) to depress the TLR4/MYD88 and STAT3 signals and impair either of the two signals inhibiting the VitD-dependent antimicrobial pathways in macrophages. Concurrently, human peripheral blood mononuclear cells (PBMCs) expressing high-level MIR337-3p exhibited a reduced ability of innate cell populations to mount fast-acting cellular immunity against intracellular mycobacterial infection. Furthermore, a higher expression of Mir337-3p after mycobacterial infection of mice coincided with much greater colony-forming unit (CFU) counts in lungs and even the death of infected animals, whereas Mir337-3p inhibitor treatment of infected mice reduced Mir337-3p levels and reversed Mir337-3p-mediated increases in CFU counts. Thus, TB-driven single MIR337-3p species could specifically target/impair both TLR4/MYD88 and STAT3 activation signals, inhibiting VitD-dependent antimicrobial response and fast-acting anti-TB immunity, leading to enhanced pathogenicity.

Genetics and Functional Mechanisms of STAT3 Polymorphisms in Human Tuberculosis.

Signal transducer and activator of transcription-3 (STAT3) plays an important role in biological balance. Our and others previous studies implied that STAT3 had a great effect on fast-acting innate immunity against tuberculosis (TB). We hypothesized that stat3 SNP down-regulation of STAT3 leads to a change in susceptibility to TB in humans. To test this hypothesis, we investigated STAT3 SNPs using SNP scan™ technique in a case-control study of TB patients (n = 470) and HC subjects (n = 356), and then conducted functional studies of them using cellular models. We found that SNPs in STAT3 3`-UTR of rs1053004 TT and rs1053005 AA genotypes or T-A haplotype were associated with susceptibility to TB or TB severity. While the TT/AA genotype correlated with the low constitutive expression of stat3 and IL-17A in PBMC, the variant stat3 of rs1053004-rs1053005 T-A haplotype indeed reduced stat3 expression in reporter assays. Interestingly, host PBMC expressing the rs1053005 AA genotype and low constitutive stat3 exhibited the reduced ability to mount fast-acting innate immunity against mycobacterial infection in cellular models. Finally, mechanistic experiments showed that the STAT3 down-regulation broadly depressed STAT3 downstream anti-mycobacterial activities involving VDR-related CAMP pathway as well as IL-32, iNOS and autophagy mechanisms, leading to an enhanced mycobacterial infection. The findings of this study suggest that low constitutive stat3 derived from the TT/AA genotype/T-A haplotype acts to down-regulate STAT3, depressing multiple anti-mycobacterial pathways/mechanisms downstream, which leads to an enhanced mycobacterial infection or TB in high-risk individuals.

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.

Circular RNA TRAPPC6B inhibits intracellular Mycobacterium tuberculosis growth while inducing autophagy in macrophages by targeting microRNA-874-3p.

OBJECTIVES: Genetic and epigenetic mechanisms regulate antimicrobial immunity against Mycobacterium tuberculosis (Mtb) infection. METHODS: The present study assessed circular RNA TRAPPC6B (circTRAPPC6B) for antimicrobial immune functions and defined mechanisms wherein circTRAPPC6B regulates Mtb growth, autophagy and microRNA in macrophages. RESULTS: The Mtb infection of monocytes/macrophages resulted in a significantly decreased level of circTRAPPC6B that inhibited intracellular Mtb growth in macrophages. Conversely, circTRAPPC6B expression enhanced autophagy or autophagy-associated protein LC3-II production in Mtb-infected macrophages. circTRAPPC6B-enhanced autophagy aggregation or sequestration was also observed in fluorescence in situ hybridisation (FISH) analysis and confocal imaging. Mechanistically, circTRAPPC6B targets an inhibiting element miR-874-3p, as shown by bioinformatics, dual-luciferase reporter gene analysis and pull-down assay, respectively. Notably, miR-874-3p prohibited autophagy via suppressing autophagy protein ATG16L1 by binding to its 3'-untranslated region (UTR) in Mtb-infected macrophages and thus promoting intracellular Mtb growth. Concurrently, circTRAPPC6B enhanced autophagy in Mtb-infected macrophages by blocking the ability of miR-874-3p to inhibit ATG16L1. Thus, circTRAPPC6B antagonises the ability of miR-874-3p to suppress ATG16L1 expression and activate and enhance autophagy sequestration to restrict Mtb growth in macrophages. CONCLUSION: The current findings suggested that both circTRAPPC6B and miR-874-3p mechanisms can be explored as potential therapeutics against Mtb infection.

Construction of Lycetin Nanocarriers and Its Effect on the Proliferation and Apoptosis of Hepatocellular Carcinoma Cells by Regulating Nuclear Factor E2 Related Factor/Antioxidant Response Element Pathway.

This article explores the role of lysin nanocarriers in inducing apoptosis of human hepatocellular carcinoma cells and the possible molecular mechanisms. Cytotoxicity tests were performed in human fibroblast cell line MRC-5. Anti-cancer activity was tested in liver cancer cell lines HepG2 and HCCLM3. The results show that nanocarriers have a targeting effect on cancer cells, have high safety, and are good delivery vehicles for drugs. In this paper, the stability of lycopene and its degradation in aqueous solutions at different temperatures were studied, and the structure and mechanism of degradation products were determined. A new type of mesoporous silica nanocarrier was synthesized as a delivery carrier of lysin and its derivatives, which has a targeting effect on cancer cells and has a slow-release effect. Surface modification can improve circulation time and stability for future resistance in vivo. The cancer experiment laid the foundation. The results showed that the lysin nanocarriers inhibited the proliferation of HepG2 and HCCLM3 human liver cancer cells in a dependent manner. After the lysin nanocarriers acted on HepG2 human hepatocellular carcinoma cells for 48 h, the cell apoptosis rate was significantly increased by flow cytometry analysis. The carrier can significantly increase the levels of reactive oxygen species and malondialdehyde, and reduce the content of reduced glutathione and superoxide dismutase. At the same time, the lysin nanocarrier can down-regulate the expression of Nrf2 and HO-1 proteins, and inhibit the occurrence of Nrf2 Nuclear displacement. The lycopene nanocarrier inhibits the proliferation of HepG2, HCCLM3 human liver cancer cells, induces apoptosis, regulates the oxidative stress response in the cell, and regulates the Nrf2/AREE antioxidant signaling pathway, thereby promoting tumor cell apoptosis.