Differential regulation of transcription factor T-bet induction during NK cell development and T helper-1 cell differentiation.

Adaptive CD4+ T helper cells and their innate counterparts, innate lymphoid cells, utilize an identical set of transcription factors (TFs) for their differentiation and functions. However, similarities and differences in the induction of these TFs in related lymphocytes are still elusive. Here, we show that T helper-1 (Th1) cells and natural killer (NK) cells displayed distinct epigenomes at the Tbx21 locus, which encodes T-bet, a critical TF for regulating type 1 immune responses. The initial induction of T-bet in NK precursors was dependent on the NK-specific DNase I hypersensitive site Tbx21-CNS-3, and the expression of the interleukin-18 (IL-18) receptor; IL-18 induced T-bet expression through the transcription factor RUNX3, which bound to Tbx21-CNS-3. By contrast, signal transducer and activator of transcription (STAT)-binding motifs within Tbx21-CNS-12 were critical for IL-12-induced T-bet expression during Th1 cell differentiation both in vitro and in vivo. Thus, type 1 innate and adaptive lymphocytes utilize distinct enhancer elements for their development and differentiation.

Transcription factors TCF-1 and GATA3 are key factors for the epigenetic priming of early innate lymphoid progenitors toward distinct cell fates.

The differentiation of innate lymphoid cells (ILCs) from hematopoietic stem cells needs to go through several multipotent progenitor stages. However, it remains unclear whether the fates of multipotent progenitors are predefined by epigenetic states. Here, we report the identification of distinct accessible chromatin regions in all lymphoid progenitors (ALPs), EILPs, and ILC precursors (ILCPs). Single-cell MNase-seq analyses revealed that EILPs contained distinct subpopulations epigenetically primed toward either dendritic cell lineages or ILC lineages. We found that TCF-1 and GATA3 co-bound to the lineage-defining sites for ILCs (LDS-Is), whereas PU.1 binding was enriched in the LDSs for alternative dendritic cells (LDS-As). TCF-1 and GATA3 were indispensable for the epigenetic priming of LDSs at the EILP stage. Our results suggest that the multipotency of progenitor cells is defined by the existence of a heterogeneous population of cells epigenetically primed for distinct downstream lineages, which are regulated by key transcription factors.

Differential Expression of the Transcription Factor GATA3 Specifies Lineage and Functions of Innate Lymphoid Cells.

Lymphoid tissue inducer (LTi) cells are regarded as a subset of innate lymphoid cells (ILCs). However, these cells are not derived from the ILC common progenitor, which generates other ILC subsets and is defined by the expression of the transcription factor PLZF. Here, we examined transcription factor(s) determining the fate of LTi progenitors versus non-LTi ILC progenitors. Conditional deletion of Gata3 resulted in the loss of PLZF+ non-LTi progenitors but not the LTi progenitors that expressed the transcription factor RORγt. Consistently, PLZF+ non-LTi progenitors expressed high amounts of GATA3, whereas GATA3 expression was low in RORγt+ LTi progenitors. The generation of both progenitors required the transcriptional regulator Id2, which defines the common helper-like innate lymphoid progenitor (ChILP), but not cytokine signaling. Nevertheless, low GATA3 expression was necessary for the generation of functionally mature LTi cells. Thus, differential expression of GATA3 determines the fates and functions of distinct ILC progenitors.

Bile Acids Control Inflammation and Metabolic Disorder through Inhibition of NLRP3 Inflammasome.

Reciprocal interactions between the metabolic system and immune cells play pivotal roles in diverse inflammatory diseases, but the underlying mechanisms remain elusive. The activation of bile acid-mediated signaling has been linked to improvement in metabolic syndromes and enhanced control of inflammation. Here, we demonstrated that bile acids inhibited NLRP3 inflammasome activation via the TGR5-cAMP-PKA axis. TGR5 bile acid receptor-induced PKA kinase activation led to the ubiquitination of NLRP3, which was associated with the PKA-induced phosphorylation of NLRP3 on a single residue, Ser 291. Furthermore, this PKA-induced phosphorylation of NLRP3 served as a critical brake on NLRP3 inflammasome activation. In addition, in vivo results indicated that bile acids and TGR5 activation blocked NLRP3 inflammasome-dependent inflammation, including lipopolysaccharide-induced systemic inflammation, alum-induced peritoneal inflammation, and type-2 diabetes-related inflammation. Altogether, our study unveils the PKA-induced phosphorylation and ubiquitination of NLRP3 and suggests TGR5 as a potential target for the treatment of NLRP3 inflammasome-related diseases.

Tespa1 is involved in late thymocyte development through the regulation of TCR-mediated signaling.

Signaling via the T cell antigen receptor (TCR) during the CD4(+)CD8(+) double-positive developmental stage determines thymocyte selection and lineage commitment. Here we describe a previously uncharacterized T cell-expressed protein, Tespa1, with critical functions during the positive selection of thymocytes. Tespa1(-/-) mice had fewer mature thymic CD4(+) and CD8(+) T cells, which reflected impaired thymocyte development. Tespa1 associated with the TCR signaling components PLC-γ1 and Grb2, and Tespa1 deficiency resulted in attenuated TCR signaling, as reflected by defective activation of the Erk-AP-1 and Ca(2+)-NFAT pathways. Our findings demonstrate that Tespa1 is a component of the TCR signalosome and is essential for T cell selection and maturation through the regulation of TCR signaling during T cell development.

B cell residency but not T cell-independent IgA switching in the gut requires innate lymphoid cells.

Immunoglobulin A (IgA)-producing plasma cells derived from conventional B cells in the gut play an important role in maintaining the homeostasis of gut flora. Both T cell-dependent and T cell-independent IgA class switching occurs in the lymphoid structures in the gut, whose formation depends on lymphoid tissue inducers (LTis), a subset of innate lymphoid cells (ILCs). However, our knowledge on the functions of non-LTi helper-like ILCs, the innate counter parts of CD4 T helper cells, in promoting IgA production is still limited. By cell adoptive transfer and utilizing a unique mouse strain, we demonstrated that the generation of IgA-producing plasma cells from B cells in the gut occurred efficiently in the absence of both T cells and helper-like ILCs and without engaging TGF-ß signaling. Nevertheless, B cell recruitment and/or retention in the gut required functional NKp46-CCR6+ LTis. Therefore, while CCR6+ LTis contribute to the accumulation of B cells in the gut through inducing lymphoid structure formation, helper-like ILCs are not essential for the T cell-independent generation of IgA-producing plasma cells.

Association of preoperative frailty with pulmonary complications after cardiac surgery in elderly individuals: a prospective cohort study.

BACKGROUND: The relationship between preoperative frailty and pulmonary complications after cardiac surgery in elderly patients is unclear. This study was designed to evaluate the relationship between frailty and postoperative pulmonary complications (PPCs) in elderly patients undergoing cardiac surgery and to provide a basis for their prevention and treatment. AIMS: This study aimed to investigate the predictive value of preoperative frailty on pulmonary complications after cardiac surgery in elderly patients. METHODS: Frailty was assessed using the CAF. The diagnosis of PPCs was based on the criteria defined by Hulzebos et al., and patients were classified into a PPCs group and a non-PPCs group. Factors with clinical significance and P < 0.05 in univariate regression analysis were included in multivariate logistic regression analysis to determine the relationship between preoperative frailty and PPCs. The area under the receiver operating characteristic (ROC) curve (AUC) was used to compare the predictive effects of the CAF, EuroSCORE II, and ASA + age on the occurrence of PPCs. RESULTS: A total of 205 patients were enrolled in this study, 31.7% of whom developed PPCs. Univariate logistic regression analysis showed that frailty, ASA grade, EuroSCORE II, hemoglobin concentration, FVC, time of operation, and postoperative AKI were associated with the development of PPCs. However, after adjustments for all possible confounding factors, multivariate logistic regression results showed that frailty, prolonged operation time, and postoperative AKI were risk factors for PPCs, and the risk of postoperative PPCs in frail patients was approximately 4.37 times that in nonfrail patients (OR = 4.37, 95%CI: 1.6-11.94, P < 0.05). The predictive efficacy of the traditional perioperative risk assessment tools EuroSCORE II and ASA + age was lower than that of CAF. CONCLUSIONS: Frailty before surgery, prolonged operation time, and postoperative AKI were independent risk factors for pulmonary complications after heart surgery in elderly individuals, and CAF was more effective than the traditional risk predictors EuroSCORE II and ASA + age.

Overexpression of the SARS-CoV-2 receptor ACE2 is induced by cigarette smoke in bronchial and alveolar epithelia.

Angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and a target for disease prevention. However, the relationship between ACE2 expression and its clinical implications in SARS-CoV-2 pathogenesis remains unknown. Here, we explored the location and expression of ACE2, and its correlation with gender, age, and cigarette smoke (CS), in a CS-exposed mouse model and 224 non-malignant lung tissues (125 non-smokers, 81 current smokers, and 18 ex-smokers) by immunohistochemistry. Moreover, the correlations of ACE2 with CS-induced oxidative stress-related markers, hypoxia-inducible factor-1α (HIF-1α), inducible nitric oxide synthase (iNOS), and 4-hydroxynonenal (4-HNE) were investigated. Chromatin immunoprecipitation and luciferase reporter assays identified the cause of ACE2 overexpression in human primary lung epithelial cells. We demonstrated that ACE2 was predominantly overexpressed on the apical surface of bronchial epithelium, while reduced in alveolar epithelium, owing to the dramatically decreased abundance of alveolar type II pneumocytes in CS-exposed mouse lungs. Consistent with this, ACE2 was primarily significantly overexpressed in human bronchial and alveolar epithelial cells in smokers regardless of age or gender. Decreased ACE2 expression was observed in bronchial epithelial cells from ex-smokers compared with current smokers, especially in those who had ceased smoking for more than 10 years. Moreover, ACE2 expression was positively correlated with the levels of HIF-1α, iNOS, and 4-HNE in both mouse and human bronchioles. The results were further validated using a publicly available dataset from The Cancer Genome Atlas (TCGA) and our previous integrated data from Affymetrix U133 Plus 2.0 microarray (AE-meta). Finally, our results showed that HIF-1α transcriptionally upregulates ACE2 expression. Our results indicate that smoking-induced ACE2 overexpression in the apical surface of bronchial epithelial cells provides a route by which SARS-CoV-2 enters host cells, which supports clinical relevance in attenuating the potential transmission risk of COVID-19 in smoking populations by smoking cessation. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Phosphatase PP2A is essential for TH17 differentiation.

Phosphatase PP2A expression levels are positively correlated to the clinical severity of systemic lupus erythematosus (SLE) and IL17A cytokine overproduction, indicating a potential role of PP2A in controlling TH17 differentiation and inflammation. By generating a mouse strain with ablation of the catalytic subunit α of PP2A in peripheral mature T cells (PP2A cKO), we demonstrate that the PP2A complex is essential for TH17 differentiation. These PP2A cKO mice had reduced TH17 cell numbers and less severe disease in an experimental autoimmune encephalomyelitis (EAE) model. PP2A deficiency also ablated C-terminal phosphorylation of SMAD2 but increased C-terminal phosphorylation of SMAD3. By regulating the activity of RORγt via binding, the changes in the phosphorylation status of these R-SMADs reduced Il17a gene transcription. Finally, PP2A inhibitors showed similar effects on TH17 cells as were observed in PP2A cKO mice, i.e., decreased TH17 differentiation and relative protection of mice from EAE. Taken together, these data demonstrate that phosphatase PP2A is essential for TH17 differentiation and that inhibition of PP2A could be a possible therapeutic approach to controlling TH17-driven autoimmune diseases.

Protein phosphatase 2A has an essential role in promoting thymocyte survival during selection.

The development of thymocytes to mature T cells in the thymus is tightly controlled by cellular selection, in which only a small fraction of thymocytes equipped with proper quality of TCRs progress to maturation. It is pivotal to protect the survival of the few T cells, which pass the selection. However, the signaling events, which safeguard the cell survival in thymus, are not totally understood. In this study, protein Ser/Thr phosphorylation in thymocytes undergoing positive selection is profiled by mass spectrometry. The results revealed large numbers of dephosphorylation changes upon T cell receptor (TCR) activation during positive selection. Subsequent substrate analysis pinpointed protein phosphatase 2A (PP2A) as the enzyme responsible for the dephosphorylation changes in developing thymocytes. PP2A catalytic subunit α (Ppp2ca) deletion in the T cell lineage in Ppp2caflox/flox-Lck-Cre mice (PP2A cKO) displayed dysregulated dephosphorylation of apoptosis-related proteins in double-positive (DP) cells and caused substantially decreased numbers of DP CD4+ CD8+ cells. Increased levels of apoptosis in PP2A cKO DP cells were found to underlie aberrant thymocyte development. Finally, the defective thymocyte development in PP2A cKO mice could be rescued by either Bcl2 transgene expression or by p53 knockout. In summary, our work reveals an essential role of PP2A in promoting thymocyte development through the regulation of cell survival.

Innate Lymphoid Cells and Intestinal Inflammatory Disorders.

Innate lymphoid cells (ILCs) are a population of lymphoid cells that do not express T cell or B cell antigen-specific receptors. They are largely tissue-resident and enriched at mucosal sites to play a protective role against pathogens. ILCs mimic the functions of CD4 T helper (Th) subsets. Type 1 innate lymphoid cells (ILC1s) are defined by the expression of signature cytokine IFN-γ and the master transcription factor T-bet, involving in the type 1 immune response; ILC2s are characterized by the expression of signature cytokine IL-5/IL-13 and the master transcription factor GATA3, participating in the type 2 immune response; ILC3s are RORγt-expressing cells and are capable of producing IL-22 and IL-17 to maintain intestinal homeostasis. The discovery and investigation of ILCs over the past decades extends our knowledge beyond classical adaptive and innate immunology. In this review, we will focus on the roles of ILCs in intestinal inflammation and related disorders.

Effects of Ultra-High Pressure on Endogenous Enzyme Activities, Protein Properties, and Quality Characteristics of Shrimp (Litopenaeus vannamei) during Iced Storage.

The present study aimed to explore the effects of ultra-high pressure (UHP) on the cathepsin (B, D, H, and L) activities, protein oxidation, and degradation properties as well as quality characteristics of iced shrimp (Litopenaeus vannamei). Fresh shrimps were vacuum-packed, treated with UHP (100-500 MPa for 5 min), and stored at 0 °C for 15 days. The results showed that the L* (luminance), b* (yellowness), W (whiteness), ΔE (color difference), hardness, shear force, gumminess, chewiness, and resilience of shrimp were significantly improved by UHP treatment. Moreover, the contents of surface hydrophobicity, myofibril fragmentation index (MFI), trichloroacetic acid (TCA)-soluble peptides, carbonyl, dityrosine, and free sulfhydryl of myofibrillar protein (MP) were significantly promoted by UHP treatment. In addition, UHP (above 300 MPa) treatment enhanced the mitochondrial membrane permeability but inhibited the lysosomal membrane stability, and the cathepsin (B, D, H, and L) activities. UHP treatment notably inhibited the activities of cathepsins, delayed protein oxidation and degradation, as well as texture softening of shrimp during storage. Generally, UHP treatment at 300 MPa for 5 min effectively delayed the protein and quality deterioration caused by endogenous enzymes and prolonged the shelf life of shrimp by 8 days.

Biocontrol Potential of Bacillus amyloliquefaciens LYZ69 Against Anthracnose of Alfalfa (Medicago sativa).

Anthracnose is a destructive disease of alfalfa (Medicago sativa) that causes severe yield losses. Biological control can be an effective and eco-friendly approach to control this alfalfa disease. In the present study, Bacillus amyloliquefaciens LYZ69, previously isolated from healthy alfalfa roots, showed a strong in vitro antifungal activity against Colletotrichum truncatum, an important causal agent of anthracnose of alfalfa. The strain LYZ69 protected alfalfa plants (biocontrol efficacy of 82.59%) from anthracnose under greenhouse conditions. The cell-free culture (CFC) of LYZ69 (20%, vol/vol) caused 60 and 100% inhibition of mycelial growth and conidial germination, respectively. High-performance liquid chromatography tandem mass spectrometry separated and identified cyclic lipopeptides (LPs) such as bacillomycin D and fengycin in the CFC of LYZ69. Light microscopy and scanning electron microscopy revealed that the mixture of cyclic LPs produced by LYZ69 caused drastic changes in mycelial morphology. Fluorescence microscopy showed that the LPs induced reactive oxygen species accumulation and caused apoptosis-like cell death in C. truncatum hyphae. In summary, our findings provide evidence to support B. amyloliquefaciens LYZ69 as a promising candidate for the biological control of anthracnose in alfalfa.

Tespa1 plays a role in the modulation of airway hyperreactivity through the IL-4/STAT6 pathway.

BACKGROUND: Thymocyte-expressed, positive selection-associated 1 (Tespa1) is a critical signaling molecule in thymocyte development. This study aimed to investigate the regulatory effect of Tespa1 on mast cells in the pathogenesis of asthma and its relationship with the interleukin (IL)-4/signal transducers and activators of transcription 6 (STAT6) signaling pathway. METHODS: Tespa1 mRNA expression analysis and IgE levels were carried out using the induced sputum of 33 adults with stable asthma and 36 healthy controls. Tespa1-knockout mice (Tespa1-/-, KO) and C57BL/6 background (wild-type, WT) mice were sensitized and treated with ovalbumin (OVA) to establish an asthma model. Pathological changes, number and activity of mast cells, and changes in activation of the IL-4/STAT6 pathway in lung tissue were detected. The changes of tryptase expression and STAT6 activation after mast cell gene knockout were analyzed in vitro. The changes of enzyme expression and STAT6 activation after mast cell gene knockout were analyzed in vitro. The association between the Tespa1 and p-STAT6 was analyzed by co-immunoprecipitation method. RESULTS: Compared with the healthy controls, Tespa1 expression was decreased, and IgE levels were elevated in the sputum of asthmatic patients. Animal experiments showed that Tespa1-/- mice exhibited more severe inflammation, higher quantity of goblet cells and mast cells in the bronchium, and greater expression of mast cell tryptase, which is induced by ovalbumin, than WT mice. And IL-4, IL-13, phospho-Janus kinase 1, and p-STAT6 expressions presented a higher increase in the Tespa1-/- mouse model than in the WT mouse model. Further in vitro studies confirmed that IL-4 could more significantly promote tryptase and p-STAT6 activities in Tespa1-/- mast cells than their WT counterparts. Correlation analysis results showed a negative correlation between Tespa1 and p-STAT6. Co-immunoprecipitation results demonstrated an association between Tespa1 and p-STAT6. CONCLUSIONS: Altogether, our results indicate that Tespa1 can negatively regulate mast cell activity, and this event is related to the mast cell IL-4/STAT6 signaling pathway and could be therapeutically exploited to treat asthma attacks.

Enzymatic transformation of ginsenosides Re, Rg1, and Rf to ginsenosides Rg2 and aglycon PPT by using ß-glucosidase from Thermotoga neapolitana.

OBJECTIVES: To enzymatically transform protopanaxatriol by using ß-glucosidase from Thermotoga neapolitana (T. neapolitana) DSM 4359. RESULTS: Recombinant ß-glucosidase was purified, which molecular weight was about 79.5 kDa. High levels of ginsenoside were obtained using the follow reaction conditions: 2 mg ml-1 ginsenoside, 25 U ml-1 enzyme, 85 °C, and pH 5.0. ß-glucosidase converted ginsenoside Re to Rg2, Rf and Rg1 to APPT completely after 3 h under the given conditions, respectively. The enzyme created 1.66 mg ml-1 Rg2 from Re with 553 mg l-1 h-1, 0.85 mg ml-1, and 1.01 mg ml-1 APPT from Rg1 and Rf with 283 and 316 mg l-1 h-1 APPT. CONCLUSIONS: ß-glucosidase could be useful for the high-yield, rapid, and low-cost preparation of ginsenoside Rg2 from Re, and APPT from the ginsenosides Rg1 and Rf.

Effect of fermentation on water mobility and distribution in fermented cornmeal using LF-NMR and its correlation with substrate.

Cordyceps militaris (C. militaris) was utilized to ferment cornmeal by solid state fermentation. The main objective of this study was to investigate effect of fermentation on the dynamic state of water and microstructure distribution of water within cornmeal with Low-field nuclear magnetic resonance, as well as the effect on composition and microstructure properties. The spin-spin relaxation time (T2) showed significant changes in solid-state fermented cornmeal. Principal component analysis further revealed that the variations within different fermentation stage could be discriminated by the T2 parameters. Bulk water (T22) was the main form of water present and lost in substrates. The weights of different indicators, as assessed by multiple regression analysis, demonstrated that there was a strong correlation between starch and T2 relaxation. Scanning electron microscopy demonstrated that fermentation can cause the appearance of micropores. The longer relaxation time of T22 during logarithmic period can be interpreted as a loosening of the structure at the starch hydrolysis, introducing more water into the structure. Thus, the differences in composition and structure of the substrate at different fermentation time produce different T2 values.

Computational study on substrate specificity of a novel cysteine protease 1 precursor from Zea mays.

Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand-enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity.

Association of point-of-care lung ultrasound findings with 30-day pulmonary complications after cardiac surgery: A prospective cohort study.

Background: Several studies have shown that bedside lung ultrasound findings in postanaesthesia care units (PACUs) and intensive care units (ICUs) correlate with postoperative pulmonary complications(PPCs) after noncardiac major surgery. However, it remains unclear whether lung ultrasound findings can be used as early predictors of PPCs in patients undergoing cardiac surgery. The main aim of our study was to evaluate the relationship between early postoperative point-of-care lung ultrasound findings and PPCs after cardiac surgery. Methods: Two board-certified physicians performed a point-of-care pulmonary ultrasound on cardiac surgery patients approximately 2 h after the patient was admitted to the ICU. Pulmonary complications occurring within 30 days postoperatively were recorded. Logistic regression modeling was used to analyze the relationship between lung ultrasound findings and PPCs. Results: PPCs occurred in 61 (30.9 %) of the 197 patients. Lung ultrasound scores(LUS), number of lung consolidation(NLC), and depth of pleural effusion(DPE) were more significant in patients who developed PPCs (P < 0.001). According to the multivariate analysis, NLC≥3(aOR 2.71,95%CI 1.14-6.44; p = 0.024)and DPE >0.95(aOR 3.79,95%CI 1.60-8.99; p = 0.002) were found to be independently associated with PPCs during this study. Conclusions: Our study demonstrated that DPE >0.95 and NLC ≥3 were associated with PPCs after cardiac surgery based on bedside lung ultrasound findings in the ICU. When these signs manifest perioperatively, the surgeon should be alerted and the necessary steps should be taken, especially if they present simultaneously.

Postharvest ripening of newly harvested corn: Weakened interactions between starch and protein.

The effects of postharvest ripening of corn on the mechanisms of starch and protein interactions were investigated using molecular dynamics and several chemical substances. Sodium dodecyl sulfate (SDS) treatment all significantly affected the starch content, molecular weight of proteins, relative crystallinity, pasting characteristics and dynamic viscoelasticity in samples before and after postharvest ripening. In the corn that had not undergone postharvest ripening, there were also significant electrostatic interactions and hydrogen bonds between starch and protein. In addition, molecular dynamics had demonstrated that the forces between starch and protein in corn were mainly hydrophobic interactions, electrostatic interaction, and hydrogen bonds. Compared with zein, corn glutelin was more tightly bound to starch. The binding energy of starch to both proteins was reduced in after postharvest-ripened corn. This study laid a rationale for investigating the change mechanism of corn postharvest ripening quality and improving processing property and edible quality of corn.