Pseudomonas aeruginosa two-component system CprRS regulates HigBA expression and bacterial cytotoxicity in response to LL-37 stress.

Pseudomonas aeruginosa is a highly pathogenic bacterium known for its ability to sense and coordinate the production of virulence factors in response to host immune responses. However, the regulatory mechanisms underlying this process have remained largely elusive. In this study, we investigate the two-component system CprRS in P. aeruginosa and unveil the crucial role of the sensor protein CprS in sensing the human host defense peptide LL-37, thereby modulating bacterial virulence. We demonstrate that CprS acts as a phosphatase in the presence of LL-37, leading to the phosphorylation and activation of the response regulator CprR. The results prove that CprR directly recognizes a specific sequence within the promoter region of the HigBA toxin-antitoxin system, resulting in enhanced expression of the toxin HigB. Importantly, LL-37-induced HigB expression promotes the production of type III secretion system effectors, leading to reduced expression of proinflammatory cytokines and increased cytotoxicity towards macrophages. Moreover, mutations in cprS or cprR significantly impair bacterial survival in both macrophage and insect infection models. This study uncovers the regulatory mechanism of the CprRS system, enabling P. aeruginosa to detect and respond to human innate immune responses while maintaining a balanced virulence gene expression profile. Additionally, this study provides new evidence and insights into the complex regulatory system of T3SS in P. aeruginosa within the host environment, contributing to a better understanding of host-microbe communication and the development of novel strategies to combat bacterial infections.

Group 1 innate lymphoid cell activation via recognition of NKG2D and liver resident macrophage MULT-1: Collaborated roles in triptolide induced hepatic immunotoxicity in mice.

Triptolide (TP) is the major bioactive component of traditional Chinese medicine Tripterygium wilfordii Hook. F., a traditional Chinese medicinal plant categorized within the Tripterygium genus of the Celastraceae family. It is recognized for its therapeutic potential in addressing a multitude of diseases. Nonetheless, TP is known to exhibit multi-organ toxicity, notably hepatotoxicity, which poses a significant concern for the well-being of patients undergoing treatment. The precise mechanisms responsible for TP-induced hepatotoxicity remain unresolved. In our previous investigation, it was determined that TP induces heightened hepatic responsiveness to exogenous lipopolysaccharide (LPS). Additionally, natural killer (NK) cells were identified as a crucial effector responsible for mediating hepatocellular damage in this context. However, associated activating receptors and the underlying mechanisms governing NK cell represented innate lymphoid cell (ILC) activation remained subjects of inquiry and were not yet investigated. Herein, activating receptor Killer cell lectin like receptor K1 (NKG2D) of group 1 ILCs was specifically upregulated in TP- and LPS-induced acute liver failure (ALF), and in vivo blockade of NKG2D significantly reduced group 1 ILC mediated cytotoxicity and mitigated TP- and LPS-induced ALF. NKG2D ligand UL16-binding protein-like transcript 1 (MULT-1) was found upregulated in liver resident macrophages (LRMs) after TP administration, and LRMs did exhibit NK cell activating effect. Furthermore, M1 polarization of LRMs cells was observed, along with an elevation in intracellular tumor necrosis factor (TNF)-α levels. In vivo neutralization of TNF-α significantly alleviated TP- and LPS-induced ALF. In conclusion, the collaborative role of group 1 ILCs and LRMs in mediating hepatotoxicity was confirmed in TP- and LPS-induced ALF. TP-induced MULT-1 expression in LRMs was the crucial mechanism in the activation of group 1 ILCs via MULT-1-NKG2D signal upon LPS stimulation, emphasizing the importance of infection control after TP administration.

Activation of cDCs and iNKT cells contributes to triptolide-induced hepatotoxicity via STING signaling pathway and endoplasmic reticulum stress.

Triptolide (TP) exhibits therapeutic potential against multiple diseases. However, its application in clinics is limited by TP-induced hepatoxicity. TP can activate invariant natural killer T (iNKT) cells in the liver, shifting Th1 cytokine bias to Th2 cytokine bias. The damaging role of iNKT cells in TP-induced hepatoxicity has been established, and iNKT cell deficiency can mitigate hepatotoxicity. However, the activation of iNKT cells in vitro by TP requires the presence of antigen-presenting cells. Therefore, we hypothesized that TP could induce dendritic cells (DCs) to activate iNKT cells, thereby leading to hepatotoxicity. The hepatic conventional DCs (cDCs) exhibited immunogenic activities after TP administration, upregulating the expression of CD1d, co-stimulatory molecules, and IL-12. Neutralization with IL-12p40 antibody extenuated TP-induced hepatotoxicity and reduced iNKT cell activation, suggesting that IL-12 could cause liver injury by activating iNKT cells. TP triggered the activation and upregulation of STING signaling pathway and increased endoplasmic reticulum (ER) stress. Downregulation of STING reduced cDC immunogenicity, inhibiting the activation of iNKT cells and hepatic damage. These indicated the regulatory effects of STING pathway on cDCs and iNKT cells, and the important roles it plays in hepatoxicity. ER stress inhibitor, 4-phenylbutyrate (4-PBA), also suppressed iNKT cell activation and liver injury, which might be regulated by the STING signaling pathway. Our results demonstrated the possible mechanisms underlying TP-induced hepatoxicity, where the activation of cDCs and iNKT cells was stimulated by upregulated STING signaling and increased ER stress as a result of TP administration.

[Effect of triptolide on Th17/Treg cells in spleen].

Triptolide( TP) is isolated from the traditional Chinese medicine Tripterygium wilfordii,which exhibits notable immuneregulative effect. Th17 cells involve in inflammatory response and Treg cells contribute to immune tolerance. They both play an important role in immune response. Previous studies have investigated that TP induced hepatic Th17/Treg imbalance. However,the effect of TP on spleen Th17/Treg cells remains unclear. Therefore,the aim of present study was to investigate the effect of TP on Th17/Treg cells in spleen. In this study,the effect of TP on the proliferation of splenic lymphocyte was detected by cytotoxicity test in vitro. After different concentrations of TP( 2. 5,5,20,40 nmol·L~(-1)) were given to splenic lymphocyte,cytokines secreted from the supernatant of splenic lymphocyte were detected by cytometric bead array,and the expression of suppressor of cytokine signaling( SOCS) mRNA was detected by qRT-PCR. Female C57 BL/6 mice were continuously observed for 24 h after treatment of 500 µg·kg-1 TP. The effects of TP on the splenic tissue structure and the percentage of Th17/Treg cells were examined. The results showed that the IC50 of TP was19. 6 nmol·L~(-1) in spleen lymphocytes. TP inhibited the secretion of IL-2 and IL-10 and induced the expression of SOCS-1/3 mRNA in spleen lymphocytes at the dosage of 2. 5 and 5 nmol·L~(-1) after 24 h in vitro. Administration of TP at dosage of 500 µg·kg-1 had no significant spleen toxicity in vivo. TP treatment increased the percentage of Th17 cells after 12 h and inhibited the proportion of Treg cells after 12 and 24 h. In conclusion,TP reduced the secretion of IL-2 and IL-10 through SOCS-1/3 signaling pathway,thereby induced the percentage of Th17 cells and inhibited the percentage of Treg cells.

[Progress of effect and mechanisms of Tripterygium wilfordii on immune system].

Traditional Chinese medicine Tripterygium wilfordii Hook.f( TWHF) is a natural botanical drug in China. It has complex chemical compositions and has been used for a long history. TWHF was used as an insecticide to protect crops at early stage,and it was later found to have significant effects in the treatment of rheumatoid arthritis,attaining great concerns. With further researches,it was found that TWHF can treat various diseases in the medical field due to a variety of pharmacological activities such as anti-cancer,neuroprotection,anti-inflammatory and immune-suppressing,particularly. Multiple extracts of TWHF have unique immunosuppressive function,playing an immune role through multi-target and multi-channel,with significant effect in the treatment of autoimmune diseases. As an immune-suppressing drug,TWHF is worthy of in-depth research due to its broad application prospects. While achieving good clinical efficacy,reports about its toxic effects to multiple systems of the body are also increasing,greatly hindering its clinical application. In order to fully understand the immune-suppressing function of TWHF and reduce or avoid the occurrence of toxic and side effects,we summarized recent progress of TWHF on the immune organs,cells and factors in recent years,as well as the pharmacology and toxic effects,hoping to provide a scientific and reasonable reference for its wider use in clinical treatment.

Structural and functional characterization of itaconyl-CoA hydratase and citramalyl-CoA lyase involved in itaconate metabolism of Pseudomonas aeruginosa.

Itaconate is a key anti-inflammatory/antibacterial metabolite in pathogen-macrophage interactions that induces adaptive changes in Pseudomonas aeruginosa-exposed airways. However, the impact and mechanisms underlying itaconate metabolism remain unclear. Our study reveals that itaconate significantly upregulates the expression of pyoverdine in P. aeruginosa and enhances its tolerance to tobramycin. Notably, the enzymes responsible for efficient itaconate metabolism, PaIch and PaCcl, play crucial roles in both utilizing itaconate and clearing its toxic metabolic intermediates. By using protein crystallography and molecular dynamics simulations analyses, we have elucidated the unique catalytic center and substrate-binding pocket of PaIch, which contribute to its highly efficient catalysis. Meanwhile, analysis of PaCcl has revealed how interactions between domains regulate the conformational changes of the active sites and binding pockets, influencing the catalytic process. Overall, our research uncovers the significance and mechanisms of PaIch and PaCcl in the efficient metabolism of itaconate by P. aeruginosa.

Synthetic biology-inspired cell engineering in diagnosis, treatment, and drug development.

The fast-developing synthetic biology (SB) has provided many genetic tools to reprogram and engineer cells for improved performance, novel functions, and diverse applications. Such cell engineering resources can play a critical role in the research and development of novel therapeutics. However, there are certain limitations and challenges in applying genetically engineered cells in clinical practice. This literature review updates the recent advances in biomedical applications, including diagnosis, treatment, and drug development, of SB-inspired cell engineering. It describes technologies and relevant examples in a clinical and experimental setup that may significantly impact the biomedicine field. At last, this review concludes the results with future directions to optimize the performances of synthetic gene circuits to regulate the therapeutic activities of cell-based tools in specific diseases.

Triptolide leads to hepatic intolerance to exogenous lipopolysaccharide and natural-killer-cell mediated hepatocellular damage by inhibiting MHC class I molecules.

BACKGROUND: Tripterygium wilfordii Hook. F (TWHF) is used as a traditional Chinese medicine, called thunder god vine, based on its efficacy for treating inflammatory diseases. However, its hepatotoxicity has limited its clinical application. Triptolide (TP) is the major active and toxic component of TWHF. Previous studies reported that a toxic pretreatment dose of TP leads to hepatic intolerance to exogenous lipopolysaccharide (LPS) stimulation, and to acute liver failure, in mice, but the immune mechanisms of TP-sensitised hepatocytes and the TP-induced excessive immune response to LPS stimulation are unknown. PURPOSE: To identify both the key immune cell population and mechanism involved in TP-induced hepatic intolerance of exogenous LPS. STUDY DESIGN: In vitro and in vivo experiments were conducted to investigate the inhibitory signal of natural killer (NK) cells maintained in hepatocytes, and the ability of TP to impair that signal. METHODS: Flow cytometry was performed to determine NK cell activity and hepatocyte histocompatibility complex (MHC) class I molecules expression; the severity of liver injury was determined based on blood chemistry values, and drug- or cell-mediated hepatocellular damage, by measuring lactate dehydrogenase (LDH) release. In vivo H-2Kb transduction was carried out using an adeno-associated viral vector. RESULTS: Interferon (IFN)-γ-mediated necroptosis occurred in C57BL/6N mice treated with 500 µg TP/kg and 0.1 mg LPS/kg to induce fulminant hepatitis. Primary hepatocytes pretreated with TP were more prone to necroptosis when exposed to recombinant murine IFN-γ. In mice administered TP and LPS, the intracellular IFN-γ levels of NK cells increased significantly. Subsequent study confirmed that NK cells were activated and resulted in potent hepatocellular toxicity. In vivo and in vitro TP administration significantly inhibited MHC class I molecules in murine hepatocytes. An in vitro analysis demonstrated the susceptibility of TP-pretreated hepatocytes to NK-cell-mediated cytotoxicity, an effect that was significantly attenuated by the induction of hepatocyte MHC-I molecules by IFN-α. In vivo induction or overexpression of hepatocyte MHC-I also protected mouse liver against TP and LPS-induced injury. CONCLUSION: The TP-induced inhibition of hepatocyte MHC-I molecules expression leads to hepatic intolerance to exogenous LPS and NK-cell mediated cytotoxicity against self-hepatocytes. These findings shed light on the toxicity of traditional Chinese medicines administered for their immunomodulatory effects.

Host-derived peptide signals regulate Pseudomonas aeruginosa virulence stress via the ParRS and CprRS two-component systems.

Pseudomonas aeruginosa, a versatile bacterium, has dual significance because of its beneficial roles in environmental soil processes and its detrimental effects as a nosocomial pathogen that causes clinical infections. Understanding adaptability to environmental stress is essential. This investigation delves into the complex interplay of two-component system (TCS), specifically ParRS and CprRS, as P. aeruginosa interprets host signals and navigates stress challenges. In this study, through phenotypic and proteomic analyses, the nuanced contributions of ParRS and CprRS to the pathogenesis and resilience mechanisms were elucidated. Furthermore, the indispensable roles of the ParS and CprS extracellular sensor domains in orchestrating signal perception remain unknown. Structural revelations imply a remarkable convergence of TCS sensors in interacting with host peptides, suggesting evolutionary strategies for bacterial adaptation. This pioneering work not only established links between cationic antimicrobial peptide (CAMP) resistance-associated TCSs and virulence modulation in nosocomial bacteria, but also transcended conventional boundaries. These implications extend beyond clinical resistance, permeating into the realm of soil revitalization and environmental guardianship. As it unveils P. aeruginosa intricacies, this study assumes a mantle of guiding strategies to mitigate clinical hazards, harness environmental advantages, and propel sustainable solutions forward.

Th17/Treg imbalance mediates hepatic intolerance to exogenous lipopolysaccharide and exacerbates liver injury in triptolide induced excessive immune response.

ETHNOPHARMACOLOGICAL RELEVANCE: Triptolide (TP) is a major active ingredient and toxic component of Tripterygium wilfordii Hook F (TWHF), which exhibits multiple activities and remarkable hepatotoxicity, the latter of which limits its clinical application due to the risk of liver injury. Previous research has revealed the hepatotoxicity of TP resulting in liver hypersensitivity upon lipopolysaccharide (LPS) stimulation. However, existing research has not elucidated the potential immune mechanism such as Th17/Treg imbalance in TP-induced hepatic excessive immune response to exogenous LPS. AIM OF THE STUDY: To investigate the role of Th17/Treg imbalance in TP-induced hepatic excessive immune response to exogenous LPS. MATERIALS AND METHODS: Mice were administered with TP, LPS, neutralization antibody and small molecule inhibitor respectively. Serum transaminase level was measured to determine the severity of liver injury. Frequencies of liver Th17 and Treg cells were analyzed by flow cytometry. Serum cytokine levels were performed by ELSIA, and mRNA levels of liver cytokine were performed by qPCR. The status of neutrophil infiltration was performed by myeloperoxidase (MPO) IHC measurement. Morphological observation of liver was performed by hematoxylin and eosin (H&E) staining. RESULTS: Mice given a single intragastric dose of TP (500 µg/kg) developed lethal fulminant hepatitis following intraperitoneal injection of LPS (0.1 mg/kg), characterized by low survival rate, severe liver injury, high levels of inflammation and neutrophil infiltration. Hepatic Th17/Treg imbalance emerged together with liver injury in these mice. Neutralization of IL-17A attenuated the liver injury and ameliorated the neutrophil infiltration. The TP-induced alteration of hepatic Th17/Treg balance was closely related to the outcome of immune-mediated acute liver injury triggered by LPS. Pretreatment with the STAT3 inhibitor AG490 effectively restored Th17/Treg balance, significantly reducing the production of IL-17A and finally attenuating the degree of liver injury. CONCLUSION: Hepatic Th17/Treg imbalance not only exacerbates TP- and LPS-induced liver injury, but also serves as an indispensable part in the mechanisms of TP-induced hepatic intolerance to exogenous endotoxin.

Triptolide increases resistance to bile duct ligation-induced liver injury and fibrosis in mice by inhibiting RELB.

Cholestasis is a common, chronic liver disease that may cause fibrosis and cirrhosis. Tripterygium wilfordii Hook.f (TWHF) is a species in the Euonymus family that is commonly used as a source of medicine and food in Eastern and Southern China. Triptolide (TP) is an epoxy diterpene lactone of TWHF, as well as the main active ingredient in TWHF. Here, we used a mouse model of common bile duct ligation (BDL) cholestasis, along with cultured human intrahepatic biliary epithelial cells, to explore whether TP can relieve cholestasis. Compared with the control treatment, TP at a dose of 70 or 140 µg/kg reduced the serum levels of the liver enzymes alanine transaminase, aspartate aminotransferase, and alkaline phosphatase in mice; hematoxylin and eosin staining also showed that TP reduced necrosis in tissues. Both in vitro and in vivo analyses revealed that TP inhibited cholangiocyte proliferation by reducing the expression of RelB. Immunohistochemical staining of CK19 and Ki67, as well as measurement of Ck19 mRNA levels in hepatic tissue, revealed that TP inhibited the BDL-induced ductular reaction. Masson 3 and Sirius Red staining for hepatic hydroxyproline showed that TP alleviated BDL-induced hepatic fibrosis. Additionally, TP substantially inhibited BDL-induced hepatic inflammation. In summary, TP inhibited the BDL-induced ductular reaction by reducing the expression of RelB in cholangiocytes, thereby alleviating liver injury, fibrosis, and inflammation.

Endoscopic retrograde appendicitis therapy (ERAT) vs appendectomy for acute uncomplicated appendicitis: A prospective multicenter randomized clinical trial.

OBJECTIVE: To compare the efficacy and feasibility of endoscopic retrograde appendicitis therapy (ERAT) with appendectomy for treating acute uncomplicated appendicitis. METHODS: This was a prospective multicenter randomized trial in which consecutive patients were randomized at a ratio of 1:1 to receive either ERAT or appendectomy. The outcomes included technical success rate, procedure time, postoperative pain relief, postoperative analgesic use, time to soft diet intake, length of postoperative hospital stay, postoperative complications, and recurrence rate. RESULTS: From August 2013 to December 2015, 110 patients with acute uncomplicated appendicitis were randomized to ERAT or appendectomy. The technical success rate was 94.55% for ERAT compared with 100% for appendectomy. Recurrence of appendicitis within 3-year follow-up occurred in 8 patients following ERAT. Postoperative abdominal pain was less frequent with ERAT than with appendectomy (21.15% [11/52] vs 87.27% [48/55], P < 0.001). Soft diet intake begun earlier after ERAT than appendectomy (6 h vs 48 h, P < 0.001), and post-procedure hospital stay was shorter (3 days vs 5 days, P < 0.001), as was the use of analgesics postoperatively (9.09% vs 49.09%, P < 0.001). CONCLUSIONS: ERAT is a feasible, safe, and effective alternative approach for the management of acute uncomplicated appendicitis. Compared with appendectomy, advantages of ERAT include no skin wound, organ preservation, reduced postoperative pain, early food intake, quick recovery, fewer postoperative complications, and shorter post-procedure hospitalization. The unsolved problem related to ERAT is the recurrence of appendicitis.

The role of invariant natural killer T cells and associated immunoregulatory factors in triptolide-induced cholestatic liver injury.

Proinflammatory cytokines are potent inhibitors of bile acid nuclear receptors and transporters. Triptolide (TP), an active ingredient of Tripterygium wilfordii Hook. f., exhibits unique efficacy for autoimmune diseases and tumors. While its clinical application is greatly constrained by hepatotoxicity. Therefore, we explored the mechanism of iNKT cells and associated immunoregulators in TP-induced cholestatic liver injury. TP was administered to both female C57BL/6 mice and Jα18-/- mice. INKT cells released significantly increased Th2 cytokine IL-4 in C57BL/6 mice after TP administration. Blood biochemistry, histopathology and immunohistochemistry demonstrated that TP-induced cholestasis liver injury. In Jα18-/- mice, cholestatic liver damage was alleviated due to the upregulation of type 2 NKT cells, nuclear receptor FXR, transporter OATP1B2 and CYP450, but also the downregulation of Cxcl10, ICAM-1 and Egr-1. Above results suggested that Th2 cytokines produced by iNKT cells suppressed type 2 NKT cells and promoted the expression of immunoregulatory factors represented by CXCL10, ICAM-1 and Egr-1, which in turn affected cholestasis-related nuclear receptor, transporter and enzymes, thus aggravated cholestatic liver injury. Our research contributes to better understanding of the role of iNKT cells in TP-induced cholestatic liver injury, thereby providing potential therapeutic targets for clinical prevention and treatment.

The role of invariant natural killer T cells in experimental xenobiotic-induced cholestatic hepatotoxicity.

Inflammation, especially the release of pro-inflammatory mediators, contributes to hepatocyte injury during cholestasis. Alpha-naphthylisothiocyanate (ANIT) is widely used in rodents to mimic clinical cholestasis. Lymphocytes have been reported to exacerbate ANIT - induced hepatotoxicity. However, which cell and mechanism mediate hepatic inflammatory response and hepatocyte injury in cholestasis is still not clear. Invariant natural killer T (iNKT) cells are a unique subset of T lymphocytes which are supposed to exert immune-regulatory effect on cholestatic liver damage. In the present study, we hypothesized that iNKT cells played a role in the pathogenesis of ANIT-induced cholestatic hepatotoxicity. ANIT (50 mg/kg, intragastric gavage) was administered to male mice for 16, 48, or 72 h. We found that ANIT administration activated iNKT cells, releasing Th1 cytokine IFN-γ and Th2 cytokine IL-4. Administration of ANIT induced cholestatic liver injury, evidenced by the elevated serum ALT, AST, ALP, TBA, TG and TC levels, and significant hepatic histopathological changes. However, knockout of iNKT cell were resistant to the late development of ANIT - induced liver injury due to the reduced release of inflammatory cytokines CXCL10 and ICAM-1, as well as the down-regulation of nuclear receptor Egr1. We further revealed that the improvement of ALP in iNKT cell - deficient mice was partly associated with the up-regulation of transporter MRP2 and NTCP and bile acid metabolism enzyme CYP2B10. Collectively, these results suggested that iNKT cells aggravated ANIT-induced cholestatic liver injury by inducing inflammatory response which contributed to the understanding of the mechanisms of ANIT-induced cholestasis. More importantly, the iNKT cell regulation may promote effective measures that control cholestasis.