Syrosingopine and UK5099 synergistically suppress non-small cell lung cancer by activating the integrated stress response.

Non-small cell lung cancer (NSCLC) presents a global health challenge due to its low five-year survival rates, underscoring the need for novel therapeutic strategies. Our research explored the synergistic mechanisms of syrosingopine and UK-5099 in treating NSCLC. In vitro experiments showed that the combination of syrosingopine and UK-5099 significantly synergized to suppress NSCLC proliferation. Further experiments revealed that this combination induced cell cycle arrest and promoted apoptosis in NSCLC cells. In vivo experiments demonstrated that the combination of syrosingopine and UK-5099 markedly inhibited tumor growth. Mechanistic studies revealed that this drug combination promoted mitochondrial damage by inducing lactate accumulation and oxidative stress. Additionally, the combination triggered an integrated stress response (ISR) through the activation of heme-regulated inhibitor kinase (HRI). Importantly, our findings suggested that the synergistic suppression of NSCLC by syrosingopine and UK-5099 was dependent on ISR activation. In summary, our study proposed a promising therapeutic approach that involved the combination of Syrosingopine and UK-5099 to activate ISR, significantly hindering NSCLC growth and proliferation.

Identification and validation of key biomarkers based on RNA methylation genes in sepsis.

Background: RNA methylation is closely involved in immune regulation, but its role in sepsis remains unknown. Here, we aim to investigate the role of RNA methylation-associated genes (RMGs) in classifying and diagnosing of sepsis. Methods: Five types of RMGs (m1A, m5C, m6Am, m7G and Ψ) were used to identify sepsis subgroups based on gene expression profile data obtained from the GEO database (GSE57065, GSE65682, and GSE95233). Unsupervised clustering analysis was used to identify distinct RNA modification subtypes. The CIBERSORT, WGCNA, GO and KEGG analysis were performed to explore immune infiltration pattern and biological function of each cluster. RF, SVM, XGB, and GLM algorithm were applied to identify the diagnostic RMGs in sepsis. Finally, the expression levels of the five key RMGs were verified by collecting PBMCs from septic patients using qRT-PCR, and their diagnostic efficacy for sepsis was verified in combination with clinical data using ROC analysis. Results: Sepsis was divided into three subtypes (cluster 1 to 3). Cluster 1 highly expressed NSUN7 and TRMT6, with the characteristic of neutrophil activation and upregulation of MAPK signaling pathways. Cluster 2 highly expressed NSUN3, and was featured by the regulation of mRNA stability and amino acid metabolism. NSUN5 and NSUN6 were upregulated in cluster 3 which was involved in ribonucleoprotein complex biogenesis and carbohydrate metabolism pathways. In addition, we identified that five RMGs (NSUN7, NOP2, PUS1, PUS3 and FTO) could function as biomarkers for clinic diagnose of sepsis. For validation, we determined that the relative expressions of NSUN7, NOP2, PUS1 and PUS3 were upregulated, while FTO was downregulated in septic patients. The area under the ROC curve (AUC) of NSUN7, NOP2, PUS1, PUS3 and FTO was 0.828, 0.707, 0.846, 0.834 and 0.976, respectively. Conclusions: Our study uncovered that dysregulation of RNA methylation genes (m1A, m5C, m6Am, m7G and Ψ) was closely involved in the pathogenesis of sepsis, providing new insights into the classification of sepsis endotypes. We also revealed that five hub RMGs could function as novel diagnostic biomarkers and potential targets for treatment.

CircRNA_0075723 protects against pneumonia-induced sepsis through inhibiting macrophage pyroptosis by sponging miR-155-5p and regulating SHIP1 expression.

Introduction: Circular RNAs (circRNAs) have been linked to regulate macrophage polarization and subsequent inflammation in sepsis. However, the underlying mechanism and the function of circRNAs in macrophage pyroptosis in pneumonia-induced sepsis are still unknown. Methods: In this study, we screened the differentially expressed circRNAs among the healthy individuals, pneumonia patients without sepsis and pneumonia-induced sepsis patients in the plasma by RNA sequencing (RNA-seq). Then we evaluated macrophage pyroptosis in sepsis patients and in vitro LPS/nigericin activated THP-1 cells. The lentiviral recombinant vector for circ_0075723 overexpression (OE-circ_0075723) and circ_0075723 silence (sh-circ_0075723) were constructed and transfected into THP-1 cells to explore the potential mechanism of circ_0075723 involved in LPS/nigericin induced macrophage pyroptosis. Results: We found circ_0075723, a novel circRNA that was significantly downregulated in pneumonia-induced sepsis patients compared to pneumonia patients without sepsis and healthy individuals. Meanwhile, pneumonia-induced sepsis patients exhibited activation of NLRP3 inflammasome and production of the pyroptosis-associated pro-inflammatory cytokines IL-1ß and IL-18. circ_0075723 inhibited macrophage pyroptosis via sponging miR-155-5p which promoted SHIP1 expression directly. Besides, we found that circ_0075723 in macrophages promoted VE-cadherin expression in endothelial cells through inhibiting the release of NLRP3 inflammasome-related cytokines, IL-1ß and IL-18, and protects endothelial cell integrity. Discussion: Our findings propose a unique approach wherein circ_0075723 suppresses macrophage pyroptosis and inflammation in pneumonia-induced sepsis via sponging with miR-155-5p and promoting SHIP1 expression. These findings indicate that circRNAs could be used as possible potential diagnostic and therapeutic targets for pneumonia-induced sepsis.

Enrichment and characterization of extracellular vesicles from ex vivo one-sided human placenta perfusion.

PROBLEM: Extracellular vesicles (EVs) released by the placenta are packed with biological information and play a major role in fetomaternal communication. Here, we describe a comprehensive set-up for the enrichment and characterization of EVs from human placenta perfusion and their application in further assays. METHOD OF STUDY: Human term placentas were used for 3 h ex vivo one-sided perfusions to simulate the intervillous circulation. Thereafter, populations of small (sEVs) and large EV (lEVs) were enriched from placental perfusate via serial ultracentrifugation. Following, EV populations were characterized regarding their size, protein concentration, RNA levels, expression of surface markers as well as their uptake and miRNA transfer to recipient cells. RESULTS: The sEV and lEV fractions from an entire perfusate yielded, respectively, 294 ± 32 µg and 525 ± 96 µg of protein equivalents and 2.6 ± 0.5 µg and 3.6 ± 0.9 µg of RNA. The sEV fraction had a mean diameter of 117 ± 47 nm, and the lEV fraction presented 236 ± 54 nm. CD63 was strongly detected by dot blot in sEVs, whereas only traces of this marker were found in lEVs. Both EV fractions were positive for the trophoblast marker PLAP (placental alkaline phosphatase) and annexin A1. EV internalization in immune cells was visualized by confocal microscopy, and the transfer of placental miRNAs was detected by quantitative real-time PCR (qPCR). CONCLUSIONS: Enriched EV populations showed characteristic features of sEVs and lEVs. EV uptake and transfer of miRNAs to recipient cells demonstrated their functional integrity. Therefore, we advocate the ex vivo one-sided placenta perfusion as a robust approach for the collection of placental EVs.

The role and mechanism of vitamin D-mediated regulation of Treg/Th17 balance in recurrent pregnancy loss.

PROBLEM: Vitamin D has a pivotal role in regulating immune responses in women with recurrent pregnancy loss (RPL), but the underlying mechanism has not been completely clarified. This study aimed to determine the correlation between vitamin D and Treg/Th17 and the effects of vitamin D supplementation on Treg/Th17 balance in RPL patients. METHODS OF STUDY: The level of vitamin D was determined in women with normal pregnancy and RPL by electrochemiluminescence. The percentages of CD4+ Foxp3+ Treg, CD4+ IL-17+ Th17, and CD4+ Foxp3+ IL-17+ T cells were determined by flow cytometry before and after vitamin D supplementation. Changes about Treg/Th17 balance after culturing with active vitamin D in vitro were determined. Vitamin D metabolic activity of peripheral blood mononuclear cells was also detected by RT-PCR. RESULTS: Compared with normal pregnancy, both the level of vitamin D and the Treg/Th17 ratio were significantly decreased in women with RPL. There was a positive correlation between the level of vitamin D and the Treg/Th17 ratio in the RPL group. Within the RPL group, those who received 2 months of vitamin D supplementation showed a significantly increased Treg/Th17 ratio compared with those without vitamin D supplementation. In vitro analysis showed that adding different concentrations of active vitamin D increased the Treg/Th17 ratio, also the mRNA levels of the vitamin D receptor and the metabolic enzyme CYP24A1 increased significantly. CONCLUSION: The occurrence of RPL may be related to vitamin D insufficiency and Treg/Th17 imbalance. The Treg/Th17 imbalance seen in women with RPL can be restored by vitamin D supplementation both in vivo and in vitro. The effects of vitamin D on the immune regulation of RPL indicate that vitamin D might be used as an alternative therapy in the future.

The role of the PD-1/PD-L1 axis in macrophage differentiation and function during pregnancy.

STUDY QUESTION: What is the role of the programmed cell death-1 (PD-1)/PD-1 ligand-1 (PD-L1) axis in macrophage polarization during early pregnancy? SUMMARY ANSWER: PD-1 signaling is a major regulator of macrophage differentiation and function, and it is critical for the success of a pregnancy. WHAT IS KNOWN ALREADY: The predominance of decidual macrophages (DMs) with an M2 phenotype is an important contributor to maternal-fetal tolerance during early pregnancy. STUDY DESIGN, SIZE, DURATION: Twenty-four women with recurrent miscarriage (RM) and 70 women undergoing elective termination of an early normal pregnancy (NP) were included. Twelve female CBA/J, four male DBA/2, and four male BALB/c mice were included and mating carried out. The 12 CBA/J pregnant mice were then categorized into three groups of four mice: healthy control group CBA/J×BALB/c, abortion-prone pregnant group CBA/J×DBA/2 and normal pregnancies CBA/J×BALB/c treated with anti-PD-1 monoclonal antibodies. PARTICIPANTS/MATERIALS, SETTING, METHODS: The profile of DMs, and the expression of PD-1 and PD-L1 in DMs from women with NP and RM were measured by flow cytometry. PD-L1 expression in human villi was determined by quantitative RT-PCR (qRT-PCR) and western blot. An in vitro model consisting of peripheral CD14+ monocytes isolated from women with NP was used. The profile of differentiated macrophages and their phagocytotic activity were then measured by flow cytometry. The mRNA levels of genes potentially underlying macrophage polarization modulated by PD-1 signaling were determined by qRT-PCR. Twelve pregnant mice were included in our in vivo model and underwent different treatment. The embryo resorption rate, and macrophage profile as well as PD-1 expression in murine spleens and uterus were analyzed by flow cytometry. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with NP, women with RM had elevated percentages of M1 DMs (P < 0.01), and reduced frequencies of M2 DMs (P < 0.05), as well as decreased PD-1 protein expression (P < 0.05) in the DMs. In addition, decreased mRNA and protein levels of PD-L1 expression in placental villi were observed in women with RM (P < 0.001). Using in vitro experiments, compared to the control group, we found that PD-1 activation by recombinant human (rh) PD-L1 Fc (human PD-L1 fused to the Fc region of human IgG1) drove the differentiation of macrophages with immuno-modulatory characteristics (P < 0.01). However, PD-1 blockade promoted dominance of the M1 phenotype (P < 0.01). PD-1 polarized macrophages showed enhanced phagocytic activity (P < 0.01), which was decreased with PD-1 blockade (P < 0.001). Furthermore, PD-1 blockade promoted the expression of pro-inflammatory cytokines and interferon regulatory factor (IRF) 5 (P < 0.05), while IRF4 expression was inhibited (P < 0.05). In addition, PD-1 blockade promoted macrophage glycolysis (P < 0.01) and inhibited fatty acid oxidation (P < 0.05). The mRNA expression levels of both phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin and mitogen-activated protein kinase/extracellular signal-regulated kinase/extracellular signal-regulated kinase were upregulated (P < 0.05) with PD-1 blockade during DM metabolic reprogramming. Moreover, in vivo mice data showed that PD-1 blockade or deficiency was associated with decreased M2 percentages at the maternal-fetal interface (P < 0.05) and embryo loss (P < 0.05). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Whether the changes in DM polarization seen in miscarriage tissues are a cause or consequence of the demise of the pregnancy still requires further investigation. In addition, conducting metabolite analysis is required to further measure bioenergetic profiles. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the role of the PD-1/PD-L1 axis in macrophage polarization during early pregnancy; such exploration enhances our understanding of the physiology of early pregnancy. Our study also indicates that targeting the PD-1 pathway may represent a novel therapeutic strategy to prevent pregnancy loss. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Nature Science Foundation of China (No. 81671490) and Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, HUST (No. 5001519002). None of the authors has any conflict of interest to declare.

The altered PD-1/PD-L1 pathway delivers the 'one-two punch' effects to promote the Treg/Th17 imbalance in pre-eclampsia.

The programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway is critical for normal pregnancy by promoting regulatory T (Treg) cell development and inhibiting the Th17 response. However, the relationship between the PD-1/PD-L1 pathway and the Treg/Th17 imbalance in pre-eclampsia (PE) is an enigma. In this study, decreased PD-1 and PD-L1 expression and a Treg/Th17 imbalance were observed at the maternal-fetal interface in PE. The regulatory effects of the PD-1/PD-L1 pathway on the Treg and Th17 cell quantities were determined in vitro by targeting T-cell proliferation, differentiation and transdifferentiation. First, decreased PD-1 expression might contribute to a higher Th17 cell frequency by promoting proliferation in PE. Second, the percentages of Treg but not Th17 cells differentiated from peripheral naive CD4+ T cells were increased by PD-L1 Fc administration. This effect was accompanied by decreased PI3K/AKT/m-TOR and increased PTEN mRNA expression and was completely reversed by PD-1 blockade. Finally, the percentage of IL-17-producing Treg cells increased and was positively associated with the Th17 cell frequency in PE. Increased RORγt and IL-17 but not Foxp3 and IL-10 mRNA expression by Treg cells was observed with PD-1 blockade. Similar findings occurred when Treg cells were exposed to IL-6/IL-23/IL-1ß and were reversed by PD-L1 Fc. Taken together, our findings indicate that the PD-1/PD-L1 pathway contributes to the Treg/Th17 imbalance via 'one-two punch' approaches: (i) promoting Th17 cell proliferation, (ii) inhibiting Treg cell differentiation and (iii) enhancing Treg cell plasticity into Th17 cells in PE. The therapeutic value of PD-L1 Fc for PE treatment will be explored in the future.

Immunological function of vitamin D during human pregnancy.

The well-established classic role of vitamin D is implicated in the regulation of the balance between calcium and phosphorus. Furthermore, vitamin D is also involved in many non-classic physiological processes, mainly including the regulation of cell proliferation, differentiation, apoptosis and immune function, participation in the inflammatory response and maintenance of genome stability function. During pregnancy, vitamin D receptor and its metabolic enzymes are expressed at the placenta and decidua, indicating the potential role in the mechanism of immunomodulation at the maternal-fetal interface. The insufficiency or deficiency of vitamin D may affect the mother directly and is related to specific pregnancy outcomes, such as preeclampsia, gestational diabetes, and recurrent miscarriage. This article reviews the effects of vitamin D on immune regulation during pregnancy.