Leucine-tRNA-synthase-2-expressing B cells contribute to colorectal cancer immunoevasion.

Immunoregulatory B cells impede antitumor immunity through unknown features and mechanisms. We report the existence of leucine-tRNA-synthase-2 (LARS2)-expressing B cell (LARS B) subset with a transforming growth factor-ß1 (TGF-ß1)-dominant regulatory feature in both mouse and human progressive colorectal cancer (CRC). Of note, LARS B cells exhibited a leucine nutrient preference and displayed active mitochondrial aminoacyl-tRNA biosynthesis. They were located outside the tertiary lymphoid structure and correlated with colorectal hyperplasia and shortened survival in CRC patients. A leucine diet induced LARS B cell generation, whereas LARS B cell deletion by Lars2 gene ablation or leucine blockage repressed CRC immunoevasion. Mechanistically, LARS2 programmed mitochondrial nicotinamide adenine dinucleotide (NAD+) regeneration and oxidative metabolism, thus determining the regulatory feature of LARS B cells in which the NAD-dependent protein deacetylase sirtuin-1 (SIRT1) was involved. We propose a leucine-dieting scheme to inhibit LARS B cells, which is safe and useful for CRC therapy.

Acetylcysteine synergizes PD-1 blockers against colorectal cancer progression by promoting TCF1+PD1+CD8+ T cell differentiation.

BACKGROUND: Programmed cell death protein 1 (PD-1) blockade is essential in treating progressive colorectal cancer (CRC). However, some patients with CRC do not respond well to immunotherapy, possibly due to the exhaustion of CD8+ T cells in the tumor microenvironment. N-Acetylcysteine (NAC) can reduce CD8+ T cell exhaustion in vitro and induce their differentiation into long-lasting phenotypes, thus enhancing the anti-tumor effect of adoptive T cell transfer. However, whether NAC can be combined with PD-1 blockade in CRC treatment and how NAC regulates CD8+ T cell differentiation remain unclear. Hence, in this study, we aimed to investigate whether NAC has a synergistic effect with PD-1 blockers against CRC progression. METHODS: We constructed a mouse CRC model to study the effect of NAC on tumors. The effect of NAC on CD8 + T cell differentiation and its potential mechanism were explored using cell flow assay and other studies in vitro and ex vivo. RESULTS: We demonstrated that NAC synergized PD-1 antibodies to inhibit CRC progression in a mouse CRC model mediated by CD8+ T cells. We further found that NAC can induce TCF1+PD1+CD8+ T cell differentiation and reduce the formation of exhausted T cells in vitro and in vivo. Moreover, NAC enhanced the expression of Glut4 in CD8+ T cells, promoting the differentiation of TCF1+PD1+CD8+ T cells. CONCLUSIONS: Our study provides a novel idea for immunotherapy for clinically progressive CRC and suggests that Glut4 may be a new immunometabolic molecular target for regulating CD8+ T cell differentiation.

HSK3486 Inhibits Colorectal Cancer Growth by Promoting Oxidative Stress and ATPase Inhibitory Factor 1 Activation.

BACKGROUND: HSK3486 (ciprofol), a new candidate drug similar to propofol, exerts sedative and hypnotic effects through gamma-aminobutyric acid type A receptors; however, its potential role in colorectal cancer is currently unknown. AIMS: This study aimed to evaluate the effects of HSK3486 on colorectal cancer cell proliferation. METHODS: Imaging was performed to detect reactive oxygen species and mitochondrial membrane potential. Western blotting was used to determine the expression of target signals. The HSK3486 molecular mechanism was investigated through ATPase inhibitory factor 1 knockdown and xenograft model experiments to assess mitochondrial function in colorectal cancer cells. RESULTS: Cell Counting Kit-8 and Annexin V/propidium iodide double staining assays showed that HSK3486 inhibited colorectal cancer cell proliferation in a concentration-dependent manner. In addition, HSK3486 treatment increased the expression of B-cell lymphoma-2-associated X, cleaved caspase 3, and cleaved poly (ADP-ribose) polymerase, whereas myeloid cell leukemia-1 and B-cell lymphoma 2 expression decreased. HSK3486 promoted mitochondrial dysfunction by inducing ATPase inhibitor factor 1 expression. Furthermore, HSK3486 promoted oxidative stress, as shown by the increase in reactive oxygen species and lactate dehydrogenase levels, along with a decrease in mitochondrial membrane potential and ATP levels. ATPase inhibitor factor 1 small interfering RNA pretreatment dramatically increased the mitochondrial membrane potential and tumor size in a xenograft model following exposure to HSK3486. CONCLUSION: Collectively, our findings revealed that HSK3486 induces oxidative stress, resulting in colorectal cancer cell apoptosis, making it a potential candidate therapeutic strategy for colorectal cancer.

Exosomal lncRNA HOTAIR induces PDL1+ B cells to impede anti-tumor immunity in colorectal cancer.

Regulatory B cells (Bregs) contribute to tumor immunosuppression. However, how B cells acquire their regulatory features in tumors remain unclear. Exosomes are important messengers that transmit tumor information to remodel tumor immunity. Here we revealed that tumor-derived exosomes drive Bregs to suppress anti-tumor immunity by delivering long non-coding RNAs (lncRNAs). HOTAIR was screened by lncRNA profiling in both colorectal cancer (CRC)-derived exosomes and infiltrating B cells. Tumor-derived HOTAIR polarized B cells toward a regulatory feature marked by programmed cell death-ligand 1 (PDL1) in CRC, and induced PDL1+ B cells to suppress CD8+ T cell activity. Exosomal HOTAIR bound to and protected pyruvate kinase M2 (PKM2) against ubiquitination degradation, resulting in STAT3 activation and PDL1 expression. Results from CRC patients showed a positive correlation between exosomal HOTAIR and tumor-infiltrating PDL1+ B cells. These findings reveal how B cells acquire PDL1-dominant regulatory feature in CRC, implying the clinical significance of exosomal therapy targeting HOTAIR.

Fasting-mimicking diet alleviates inflammatory pain by inhibiting neutrophil extracellular traps formation and neuroinflammation in the spinal cord.

BACKGROUND: Neutrophil extracellular traps (NETs) promote neuroinflammation and, thus, central nervous system (CNS) disease progression. However, it remains unclear whether CNS-associated NETs affect pain outcomes. A fasting-mimicking diet (FMD) alleviates neurological disorders by attenuating neuroinflammation and promoting nerve regeneration. Hence, in this study, we explore the role of NETs in the CNS during acute pain and investigate the role of FMD in inhibiting NETs and relieving pain. METHODS: The inflammatory pain model was established by injecting complete Freund's adjuvant (CFA) into the hind paw of mice. The FMD diet regimen was performed during the perioperative period. PAD4 siRNA or CI-amidine (PAD4 inhibitor) was used to inhibit the formation of NETs. Monoamine oxidase-B (MAO-B) knockdown occurred by AAV-GFAP-shRNA or AAV-hSyn-shRNA or was inhibited by selegiline (an MAO-B inhibitor). The changes in NETs, neuroinflammation, and related signaling pathways were examined by western blot, immunofluorescence, ELISA, and flow cytometry. RESULTS: In the acute phase of inflammatory pain, NETs accumulate in the spinal cords of mice. This is associated with exacerbated neuroinflammation. Meanwhile, inhibition of NETs formation alleviates allodynia and neuroinflammation in CFA mice. FMD inhibits NETs production and alleviates inflammatory pain, which is enhanced by treatment with the NETs inhibitor CI-amidine, and reversed by treatment with the NETs inducer phorbol 12-myristate 13-acetate (PMA). Mechanistically, the neutrophil-recruiting pathway MAO-B/5-hydroxyindoleacetic acid (5-HIAA) / G-protein-coupled receptor 35 (GPR35) and NETs-inducing pathway MAO-B/ Reactive oxygen species (ROS) are significantly upregulated during the development of inflammatory pain. MAO-B is largely expressed in astrocytes and neurons in the spinal cords of CFA mice. However, knockdown or inhibition of MAO-B effectively attenuates CFA-induced inflammatory pain, NETs formation, and neuroinflammation in the spinal cord. Moreover, within rescue experiments, MAO-B inhibitors synergistically enhance FMD-induced pain relief, NETs inhibition, and neuroinflammation attenuation, whereas supplementation with MAO-B downstream molecules (i.e., 5-HIAA and PMA) abolished this effect. CONCLUSIONS: Neutrophil-released NETs in the spinal cord contribute to pain development. FMD inhibits NETs formation and NETs-induced neuroinflammation by inhibiting the MAO-B/5-HIAA/GPR35 and MAO-B/ROS pathways in astrocytes and neurons, thereby relieving pain progression. Video Abstract.

ets1 associates with KMT5A to participate in high glucose-mediated EndMT via upregulation of PFN2 expression in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is currently the leading cause of end-stage renal disease globally. The endothelial-to-mesenchymal transition (EndMT) of glomerular endothelial cells has been reported to play a crucial role in DN. As a specific form of epithelial-to-mesenchymal transition, EndMT and epithelial-to-mesenchymal transition may exhibit mutual modulators. Profilin 2 (PFN2) has been reported to participate in epithelial-to-mesenchymal transition. Moreover, ETS proto-oncogene 1 (ets1) and lysine methyltransferase 5A (KMT5A) have been reported to contribute to high glucose-mediated endothelial injury and epithelial-to-mesenchymal transition. In this study, we hypothesize ets1 associates with KMT5A to modulate PFN2 transcription, thus participating in high glucose-mediated EndMT in glomerular endothelial cells. METHODS: Immunohistochemistry (IHC) was performed to detect protein levels in the kidney tissues and/or aorta tissues of human subjects and rats. Western blot, qPCR and immunofluorescence were performed using human umbilical vein endothelial cells (HUVECs). Chromatin immunoprecipitation (ChIP) assays and dual luciferase assays were performed to assess transcriptional activity. The difference between the groups was compared by two-tailed unpaired t-tests or one-way ANOVAs. RESULTS: Our data indicated that vimentin, αSMA, S100A4 and PFN2 levels were increased, and CD31 levels were reduced in glomerular endothelial cells of DN patients and rats. Our cell experiments showed that high glucose induced EndMT by augmenting PFN2 expression in HUVECs. Moreover, high glucose increased ets1 expression. si-ets1 suppressed high glucose-induced PFN2 levels and EndMT. ets1 overexpression-mediated EndMT was reversed by si-PFN2. Furthermore, ets1 was determined to associate with KMT5A. High glucose attenuated KMT5A levels and histone H4 lysine 20 methylation (H4K20me1), one of the downstream targets of KMT5A. KMT5A upregulation suppressed high glucose-induced PFN2 levels and EndMT. sh-KMT5A-mediated EndMT was counteracted by si-PFN2. Furthermore, H4K20me1 and ets1 occupied the PFN2 promoter region. sh-KMT5A cooperated with ets1 overexpression to activate PFN2 promoter activity. Our in vivo study demonstrated that KMT5A was reduced, while ets1 was augmented, in glomerular endothelial cells of DN patients and rats. CONCLUSIONS: The present study indicated that ets1 cooperated with KMT5A to transcribe PFN2, thus contributing to hyperglycemia-induced EndMT in the glomerular endothelial cells of DN patients and rats. Trial registration ChiCTR, ChiCTR2000029425. 2020/1/31, http://www.chictr.org.cn/showproj.aspx?proj=48548.

Fasting-Mimicking Diet Drives Antitumor Immunity against Colorectal Cancer by Reducing IgA-Producing Cells.

As a safe, feasible, and inexpensive dietary intervention, fasting-mimicking diet (FMD) exhibits excellent antitumor efficacy by regulating metabolism and boosting antitumor immunity. A better understanding of the specific mechanisms underlying the immunoregulatory functions of FMD could help improve and expand the clinical application of FMD-mediated immunotherapeutic strategies. In this study, we aimed to elucidate the role of metabolic reprogramming induced by FMD in activation of antitumor immunity against colorectal cancer. Single-cell RNA sequencing analysis of intratumoral immune cells revealed that tumor-infiltrating IgA+ B cells were significantly reduced by FMD treatment, leading to the activation of antitumor immunity and tumor regression in murine colorectal cancer models. Mechanistically, FMD delayed tumor growth by repressing B-cell class switching to IgA. Therefore, FMD-induced reduction of IgA+ B cells overcame the suppression of CD8+ T cells. The immunoregulatory and antitumor effects of FMD intervention were reversed by IgA+ B-cell transfer. Moreover, FMD boosted fatty acid oxidation (FAO) to trigger RUNX3 acetylation, thus inactivating Cα gene transcription and IgA class switching. IgA+ B-cell expansion was also impeded in patients placed on FMD, while B-cell expression of carnitine palmitoyl transferase 1A (CPT1A), the rate-limiting enzyme of FAO, was increased. Furthermore, CPT1A expression was negatively correlated with both IgA+ B cells and IgA secretion within colorectal cancer. Together, these results highlight that FMD holds great promise for treating colorectal cancer. Furthermore, the degree of IgA+ B cell infiltration and FAO-associated metabolic status are potential biomarkers for evaluating FMD efficacy. SIGNIFICANCE: Metabolic reprogramming of B cells induced by fasting-mimicking diet suppresses IgA class switching and production to activate antitumor immunity and inhibit tumor growth. See related commentary by Bush and Perry, p. 3493.

Neutrophil extracellular traps-triggered impaired autophagic flux via METTL3 underlies sepsis-associated acute lung injury.

Neutrophil extracellular traps (NETs) assist pathogen clearance, while excessive NETs formation is associated with exacerbated inflammatory responses and tissue injury in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Autophagy is generally considered to be a protective process, but autophagy dysfunction is harmful. Whether and how NETs affect autophagic flux during sepsis-induced ALI are currently unknown. Here, we confirmed that the level of NETs was increased in ARDS patients and mice models, which led to impairment of autophagic flux and deterioration of the disease. Mechanistically, NETs activated METTL3 mediated m6A methylation of Sirt1 mRNA in alveolar epithelial cells, resulting in abnormal autophagy. These findings provide new insights into how NETs contribute to the development of sepsis-associated ALI/ARDS.

mRNAsi-related metabolic risk score model identifies poor prognosis, immunoevasive contexture, and low chemotherapy response in colorectal cancer patients through machine learning.

Colorectal cancer (CRC) is one of the most fatal cancers of the digestive system. Although cancer stem cells and metabolic reprogramming have an important effect on tumor progression and drug resistance, their combined effect on CRC prognosis remains unclear. Therefore, we generated a 21-gene mRNA stemness index-related metabolic risk score model, which was examined in The Cancer Genome Atlas and Gene Expression Omnibus databases (1323 patients) and validated using the Zhongshan Hospital cohort (200 patients). The high-risk group showed more immune infiltrations; higher levels of immunosuppressive checkpoints, such as CD274, tumor mutation burden, and resistance to chemotherapeutics; potentially better response to immune therapy; worse prognosis; and advanced stage of tumor node metastasis than the low-risk group. The combination of risk score and clinical characteristics was effective in predicting overall survival. Zhongshan cohort validated that high-risk score group correlated with malignant progression, worse prognosis, inferior adjuvant chemotherapy responsiveness of CRC, and shaped an immunoevasive contexture. This tool may provide a more accurate risk stratification in CRC and screening of patients with CRC responsive to immunotherapy.

KMT5A downregulation participated in High Glucose-mediated EndMT via Upregulation of ENO1 Expression in Diabetic Nephropathy.

Diabetic nephropathy (DN) has become the common and principal microvascular complication of diabetes that could lead to end-stage renal disease. It was reported endothelial-to-mesenchymal transition (EndMT) in glomeruli plays an important role in DN. Enolase1 (ENO1) and Lysine Methyltransferase 5A (KMT5A) were found to modulate epithelial-to-mesenchymal transition in some situations. In the present study, we speculated KMT5A regulates ENO1 transcript, thus participating in hyperglycemia-induced EndMT in glomeruli of DN. Our study represented vimentin, αSMA and ENO1 expression elevated, and CD31 expression decreased in glomeruli of DN participants and rats. In vitro, high glucose induced EndMT by increase of ENO1 levels. Moreover, high glucose downregulated KMT5A levels and increased regulatory factor X1 (RFX1) levels. KMT5A upregulation or si-RFX1 decreased high glucose-induced ENO1 expression and EndMT. RFX1 overexpression- or sh-KMT5A-induced EndMT was attenuated by si-ENO1. Further, the association between KMT5A and RFX1 was verified. Furthermore, histone H4 lysine20 methylation (the direct target of KMT5A) and RFX1 positioned on ENO1 promoter region. sh-KMT5A enhanced positive action of RFX1 on ENO1 promoter activity. KMT5A reduction and RFX1 upregulation were verified in glomeruli of DN patients and rats. KMT5A associated with RFX1 to modulate ENO1, thus involved in hyperglycemia-mediated EndMT in glomeruli of DN.

Pro- and Anti- Effects of Immunoglobulin A- Producing B Cell in Tumors and Its Triggers.

B cells are well known as key mediators of humoral immune responses via the production of antibodies. Immunoglobulin A (IgA) is the most abundantly produced antibody isotype and provides the first line of immune protection at mucosal surfaces. However, IgA has long been a divisive molecule with respect to tumor progression. IgA exerts anti- or pro-tumor effect in different tumor types. In this review, we summarize emerging evidence regarding the production and effects of IgA and IgA+ cells in the tumor microenvironment (TME). Moreover, we discuss that the TME cytokines, host diet, microbiome, and metabolites play a pivotal role in controlling the class-switch recombination (CSR) of IgA. The analysis of intratumoral Ig repertoires and determination of metabolites that influence CSR may help establish novel therapeutic targets for the treatment of cancers.

Impact of anesthesia methods on perioperative systemic inflammation and long-term outcomes in patients undergoing surgery for hepatocellular carcinoma: a propensity score-matched analysis.

BACKGROUND: Recent studies have shown regional anesthesia might improve the survival of cancer patients. We hypothesized that general-epidural anesthesia (GEA) was associated with longer survival than general anesthesia (GA) in patients undergoing hepatocellular carcinoma (HCC) resections. METHODS: A retrospective study included patients who received curative resection for HCC between January 2014 to December 2017. Patients were grouped in GEA vs. GA. After propensity score matching, perioperative inflammatory scores were calculated. Grade of postoperative complications, length of stay (LOS), dosage of sufentanil used and times of patients requiring rescue analgesia in both groups were compared for intraoperative and postoperative parameter. Survival curves were constructed from the date of surgery to death, univariable and multivariable Cox regression models were used to compare hazard ratios for death. RESULTS: A total of 772 patients were included in the study. With 386 patients in GA group and 386 patients in GEA group. After propensity score matching, the demographic and baseline biomarkers in the two groups were similar. Patients in GEA group showed significantly lower inflammatory scores. Grade of postoperative complications, LOS, opioid use, and times of patients requiring rescue analgesia was significantly lower in the GEA group. The overall survival (OS) and disease-free survival (DFS) rate was significantly lower in the GA group (54.2% vs. 62.3%, 41.2% vs. 52.5%, P<0.001). The multivariate analysis indicated the GA was associated with shorter OS (HR: 1.28, 95% CI: 1.07-2.02, P<0.001) and DFS (HR: 1.06, 95% CI: 1.03-1.71, P<0.001). CONCLUSIONS: GA combined with epidural anesthesia is associated with lower levels of inflammation, and longer survival in patients undergoing hepatectomy.

Dezocine regulates the malignant potential and aerobic glycolysis of liver cancer targeting Akt1/GSK-3ß pathway.

BACKGROUND: Due to the "ceiling effect" of respiratory depression and the non-addictiveness, the consumption of dezocine is increasing quickly in the cancer surgery perioperative period for security and comfort reasons in China. Former studies find dezocine inhibits the norepinephrine transporters (NET) and serotonin transporters (SERT) and sigma-1opioid receptors. Given the complexity of the molecular mechanism, the effect of dezocine on tumor cells need to be studied. In this study, we investigated the effect of dezocine on HepG2 and Hep 3B liver cancer cell lines growth and glycolysis, and the molecular mechanisms behind. METHODS: HepG2 and Hep 3B cells viability and migration were measured by CCK8, Wound healing and transwell assay, Extracellular acidification rate (ECAR) was used to index the aerobic glycolysis of liver cancer cells and western blot analysis showed protein expression levels in the cells. SC79, an agonist of Akt, and the siRNA silence of Akt1 aimed to regulate Akt1 activity and expression in the reverse experiments. RESULTS: Dezocine played opposite roles in HepG2 and Hep 3B cells viability and migration in a concentration-dependent manner (P<0.01). Dezocine has diverse effects on aerobic glycolysis and adjusts the serine/threonine kinase 1 (Akt1)-glycogen synthase kinase-3ß (GSK-3ß) pathway. The effects of SC79 and the siRNA silence of Akt1 could reverse the effects of dezocine on HepG2 and Hep 3B cells. CONCLUSIONS: As an analgesic drug widely used in clinical practice, dezocine play reversed roles on HepG2 and Hep 3B cells viability and migration targeting Akt1/GSK-3ß pathway then the glycolysis in a concentration-dependent manner.