Single cell RNA-sequencing analysis reveals that N-acetylcysteine partially reverses hepatic immune dysfunction in biliary atresia.

Background & Aims: Our previous study indicated that CD177+ neutrophil activation has a vital role in the pathogenesis of biliary atresia (BA), which is partially ameliorated by N-acetylcysteine (NAC) treatment. Here, we evaluated the clinical efficacy of NAC treatment and profiled liver-resident immune cells via single cell RNA-sequencing (scRNA-seq) analysis to provide a comprehensive immune landscape of NAC-derived immune regulation. Methods: A pilot clinical study was conducted to evaluate the potential effects of intravenous NAC treatment on infants with BA, and a 3-month follow-up was carried out to assess treatment efficacy. scRNA-seq analysis of liver CD45+ immune cells in the control (n = 4), BA (n = 6), and BA + NAC (n = 6) groups was performed and the effects on innate cells, including neutrophil and monocyte-macrophage subsets, and lymphoid cells were evaluated. Results: Intravenous NAC treatment demonstrated beneficial efficacy for infants with BA by improving bilirubin metabolism and bile acid flow. Two hepatic neutrophil subsets of innate cells were identified by scRNA-seq analysis. NAC treatment suppressed oxidative phosphorylation and reactive oxygen species production in immature neutrophils, which were transcriptionally and functionally similar to CD177+ neutrophils. We also observed the suppression of hepatic monocyte-mediated inflammation, decreased levels of oxidative phosphorylation, and M1 polarisation in Kupffer-like macrophages by NAC. In lymphoid cells, enhancement of humoral immune responses and attenuation of cellular immune responses were observed after NAC treatment. Moreover, cell-cell interaction analysis showed that innate/adaptive proinflammatory responses were downregulated by NAC. Conclusions: Our clinical and scRNA-seq data demonstrated that intravenous NAC treatment partially reversed liver immune dysfunction, alleviated the proinflammatory responses in BA by targeting innate cells, and exhibited beneficial clinical efficacy. Impact and implications: BA is a serious liver disease that affects newborns and has no effective drug treatment. In this study, scRNA-seq showed that NAC treatment can partially reverse the immune dysfunction of neutrophil extracellular trap-releasing CD177+ neutrophils and Kupffer cells, and lower the inflammatory responses of other innate immune cells in BA. In consequence, intravenous NAC treatment improved the clinical outcomes of patients with BA in term of bilirubin metabolism.

CD177+ cells produce neutrophil extracellular traps that promote biliary atresia.

BACKGROUND & AIMS: We have previously reported on the potential pathogenic role of neutrophils in biliary atresia (BA). Herein, we aimed to delineate the role of CD177+ neutrophils in the pathogenesis of BA. METHODS: Immune cells from the livers of mice with rhesus rotavirus-induced BA were analysed. Single-cell RNA-sequencing was performed to specifically analyse Gr-1+ (Ly6C/Ly6G+) cells in the liver. Gene expression profiles of CD177+ cells were analysed using the Smart-Seq RNA-sequencing method, and the pathogenesis of BA was examined in Cd177-/- mice. Neutrophil extracellular trap (NET) inhibitors were used to determine the role of CD177+ cell-derived NETs in BA-associated bile duct damage, and a pilot clinical study evaluated the potential effects of N-acetylcysteine on NET release in BA. RESULTS: Increased levels of Gr-1+ cells were observed in the livers of mice with rhesus rotavirus-induced BA. RNA-sequencing analysis revealed that CD177+ cells were the main population of Gr-1+ cells and expressed elevated levels of both interferon-stimulated and neutrophil degranulation genes. Cd177-/- BALB/c mice exhibited delayed disease onset and reduced morbidity and mortality. High numbers of mitochondria were detected in CD177+ cells derived from mice with BA; these cells were associated with increased levels of reactive oxygen species and increased NET formation, which induced the apoptosis of biliary epithelial cells in cocultures. In a pilot clinical study, the administration of N-acetylcysteine to patients with BA reduced CD177+ cell numbers and reactive oxygen species levels, indicating a potential beneficial effect. CONCLUSIONS: Our data indicate that CD177+ cells play an important role in the initiation of BA pathogenesis via NET formation. CLINICAL TRIAL REGISTRATION: The pilot study of N-acetylcysteine treatment in patients with BA was registered on the Chinese Clinical Trial Registry (ChiCTR2000040505). LAY SUMMARY: Neutrophils (a type of innate immune cell, i.e. an immune cell that doesn't target a specific antigen) are thought to play a role in the development of biliary atresia (a rare but potentially lethal condition of the bile ducts that occurs in infants). Herein, we found that neutrophils expressing a particular protein (CD177) played an important role in bile duct damage by releasing a special structure (NET) that can trap and kill pathogens but that can also cause severe tissue damage. A pilot study in patients with biliary atresia showed that inhibiting NETs could have a beneficial effect.

Modified Fusicoccane-Type Diterpenoids from Alternaria brassicicola.

Seven new modified fusicoccane-type diterpenoids (1-7), together with two known congeners (8 and 9), were obtained from Alternaria brassicicola. Their structures were elucidated from a combination of NMR and HRESIMS data and 13C NMR calculation as well as DP4+ probability analyses, and the absolute configurations of 1-5 were determined by ECD calculation and single-crystal X-ray diffraction (Cu Kα). Compounds 1-3 belong to a rare class of 16-nor-dicyclopenta[a,d]cyclooctane diterpenoids, and compounds 2 and 4 represent the first examples of fusicoccane-type diterpenoids featuring two previously undescribed tetracyclic 5/6/6/5 ring systems, while compound 5 features a previously undescribed tetracyclic 5/8/5/3 ring system. Compound 7 was moderately anti-inflammatory, and compounds 2, 3, 5, and 7 were weakly cytotoxic.

Novel Antimicrobial Compounds as Ophiobolin-Type Sesterterpenes and Pimarane-Type Diterpene From Bipolaris Species TJ403-B1.

Six previously undescribed ophiobolin-type sesterterpenes, namely, bipolatoxins A-F (1-6); and one previously undescribed pimarane-type diterpene, namely, 1ß-hydroxy momilactone A (7); together with three known compounds, namely, 25-hydroxyophiobolin I (8), ophiobolin I (9), and ophiobolin A lactone (10); were isolated and identified from the endophytic fungus Bipolaris species TJ403-B1. Their structures with absolute configurations were elucidated on the basis of extensive spectroscopic analyses (including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy data), single-crystal X-ray diffraction analyses, and comparison of experimental circular dichroism data. All compounds (except for 5) were evaluated for antimicrobial potential, which indicated that bipolatoxin D (4) showed significant inhibitory activity against Enterococcus faecalis with a minimum inhibitory concentration (MIC) value of 8 µg/mL, and ophiobolin A lactone (10) showed significant inhibitory activity against Acinetobacter baumannii and E. faecalis with MIC values of 8 and 8 µg/mL, respectively.

Periconiastone A, an Antibacterial Ergosterol with a Pentacyclo[8.7.0.01,5.02,14.010,15]heptadecane System from Periconia sp. TJ403-rc01.

Periconiastone A (1), an ergosterol with an unprecedented pentacyclo[8.7.0.01,5.02,14.010,15]heptadecane system, was isolated from Periconia sp. TJ403-rc01. Its structure was assigned by extensive spectroscopic analyses and quantum-chemical 13C NMR and ECD calculations. A vinylogous α-ketol rearrangement and an aldol condensation reaction during biosynthesis were proposed as key steps for the formation of 1. Compound 1 showed antibacterial activity against Gram-positive S. aureus and E. faecalis with MIC values of 4 and 32 µg/mL, respectively.

Fusaresters A-E, new γ-pyrone-containing polyketides from fungus Fusarium sp. Hungcl and structure revision of fusariumin D.

Marine-derived fungi have been regarded as an under-explored and promising reservoir of structurally novel and bioactive natural products. In this study, five new γ-pyrone-containing polyketides, fusaresters A-E (1-5), were isolated and identified from the culture extracts of a marine-derived fungus Fusarium sp. Hungcl. The structures of compounds 1-5 were elucidated on the basis of their HRESIMS and NMR spectroscopic data as well as 13C NMR calculation and electronic circular dichroism (ECD) analyses. Remarkably, the structure of fusariumin D was revised to (9S*,11S*)-3. All these isolates were tested for the cytotoxicity against seven human cancer cell lines, including SW480, HL-60, A549, MCF-7, HepG2, HeLa and SMMC-7721, and the inhibitory activity against protein tyrosine phosphatase 1B (PTP1B). The results revealed that only compound 2 showed a weak inhibition rate of 56% at 40 µM.

Antibacterial activity against drug-resistant microbial pathogens of cytochalasan alkaloids from the arthropod-associated fungus Chaetomium globosum TW1-1.

By feeding 1-methyl-l-tryptophan (1-MT) into cultures of the arthropod-associated fungus Chaetomium globosum TW1-1, three novel cytochalasan alkaloids, termed as armochaetoglosins A-C (1-3), together with five known analogues, namely prochaetoglobosin I (4), chaetoglobosin T (5), chaetoglobosin C (6), armochaetoglobin Y (7), and chaetoglobosin Vb (8), were isolated and characterized. Their structures including absolute configurations were elucidated by means of NMR spectroscopy, single-crystal X-ray crystallography, and comparison of the experimental electronic circular dichroism (ECD) spectra. Structurally, compounds 1-3 represented the first examples of 1'-N-methyl-chaetoglobosins, which were possibly biosynthesized from the additive 1-MT rather than tryptophan. Additionally, compound 3 showed the highest antibacterial activity against K. pneumoniae and ESBL-E. coli with MIC values of 4.0 µg/mL and 16.0 µg/mL, respectively, wherein the inhibitory effect of 3 against K. pneumoniae was stronger than that of the clinically used antibiotic meropenem, with an MIC value of 8 µg/mL. Our findings may provide new chemical templates for the development of new antibacterial agents against drug-resistant microbial pathogens.

Armochaetoglasins A-I: Cytochalasan alkaloids from fermentation broth of Chaetomium globosum TW1-1 by feeding L-tyrosine.

By feeding L-tyrosine into the culture medium, nine undescribed compounds, termed as armochaetoglasins A-I, together with three known analogues, namely armochaetoglobin E, chaetoglobosin V, and chaetoglobosin J, were isolated and identified from the medicinal terrestrial arthropod-derived fungus Chaetomium globosum TW1-1. Their structures were elucidated by means of NMR spectroscopy, single-crystal X-ray crystallography, and comparison of their electronic circular dichroism (ECD) spectra. Structurally, armochaetoglasin A represented the first tyrosine-derived cytochalasan alkaloid characterized by a 13-membered carbocyclic ring system; armochaetoglasins B and C possessed a rare 19,20-seco-chaetoglobosin skeleton. Armochaetoglasin B, chaetoglobosin V, and chaetoglobosin J showed weak cytotoxic activity with IC50 values ranging from 19.5 to 34.72 µM.