Single-Cell Sequencing Reveals MYOF-Enriched Monocyte/Macrophage Subcluster as a Favorable Prognostic Factor in Sepsis.

This research utilized single-cell RNA sequencing to map the immune cell landscape in sepsis, revealing 28 distinct cell clusters and categorizing them into nine major types. Delving into the monocyte/macrophage subclusters, 12 unique subclusters are identified and pathway enrichment analyses are conducted using KEGG and GO, discovering enriched pathways such as oxidative phosphorylation and antigen processing. Further GSVA and AUCell assessments show varied activation of interferon pathways, especially in subclusters 4 and 11. The clinical correlation analysis reveals genes significantly linked to survival outcomes. Additionally, cellular differentiation in these subclusters is explored. Building on these insights, the differential gene expression within these subclusters is specifically scrutinized, which reveal MYOF as a key gene with elevated expression levels in the survivor group. This finding is further supported by in-depth pathway enrichment analysis and the examination of cellular differentiation trajectories, where MYOF's role became evident in the context of immune response regulation and sepsis progression. Validating the role of the MYOF gene in sepsis, a dose-dependent response to LPS in THP-1 cells and C57 mice is observed. Finally, inter-cellular communications are analyzed, particularly focusing on the MYOF+Mono/Macro subcluster, which indicates a pivotal role in immune regulation and potential therapeutic targeting.

KMT2D links TGF-ß signaling to noncanonical activin pathway and regulates pancreatic cancer cell plasticity.

Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-ß to the activin A pathway. We found that TGF-ß upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a noncanonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the protumoral role of KMT2D loss. These findings support a tumor-suppressive role of KMT2D in pancreatic cancer and identify miR-147b and activin A as novel therapeutic targets.

c-Myb Dominates TBK1-Mediated Endotoxin Tolerance in Kupffer Cells by Negatively Regulating DTX4.

As a protective mechanism regulating excessive inflammation, endotoxin tolerance plays a vital role in regulating endotoxin shock. Kupffer cells are players in mediating endotoxin tolerance. Nonetheless, the regulatory mechanism regulating endotoxin tolerance is barely known. A nonclassical IKK kinase called TRAF-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) can regulate inflammation. Here, we found that TBK1 is required for endotoxin tolerance in Kupffer cells. TBK1 plays a dominant role in regulating endotoxin tolerance by negatively regulating the induction of p100 processing. Deltex E3 ubiquitin ligase 4 (DTX4), a negative regulator of TBK1, can promote TBK1 K48-mediated ubiquitination and indirectly regulate endotoxin tolerance in Kupffer cells. We demonstrate that the c-Myb transcription factor could negatively regulate DTX4. Overexpression of c-Myb can be used to reduce the ubiquitination of TBK1 by reducing DTX4 transcription and to boost the anti-inflammatory effect of endotoxin tolerance. Thus, this study reveals a novel theory of TBK1-mediated endotoxin tolerance in Kupffer cells.

KDM6A Loss Recruits Tumor-Associated Neutrophils and Promotes Neutrophil Extracellular Trap Formation in Pancreatic Cancer.

Lysine (K)-specific demethylase 6A (KDM6A) is a frequently mutated tumor suppressor gene in pancreatic ductal adenocarcinoma (PDAC). However, the impact of KDM6A loss on the PDAC tumor immune microenvironment is not known. This study used a genetically engineered, pancreas-specific Kdm6a knockout (KO) PDAC mouse model and human PDAC tissue samples to demonstrate that KDM6A loss correlates with increased tumor-associated neutrophils and neutrophil extracellular traps (NET) formation, which are known to contribute to PDAC progression. Genome-wide bromouridine sequencing analysis to evaluate nascent RNA synthesis showed that the expression of many chemotactic cytokines, especially CXC motif chemokine ligand 1 (CXCL1), was upregulated in KDM6A KO PDAC cells. KDM6A-deficient PDAC cells secreted higher levels of CXCL1 protein, which in turn recruited neutrophils. Furthermore, in a syngeneic orthotopic mouse model, treatment with a CXCL1 neutralizing antibody blocked the chemotactic and NET-promoting properties of KDM6A-deficient PDAC cells and suppressed tumor growth, confirming CXCL1 as a key mediator of chemotaxis and PDAC growth driven by KDM6A loss. These findings shed light on how KDM6A regulates the tumor immune microenvironment and PDAC progression and suggests that the CXCL1-CXCR2 axis may be a candidate target in PDAC with KDM6A loss. SIGNIFICANCE: KDM6A loss in pancreatic cancer cells alters the immune microenvironment by increasing CXCL1 secretion and neutrophil recruitment, providing a rationale for targeting the CXCL1-CXCR2 signaling axis in tumors with low KDM6A.

TBK1 participates in glutaminolysis by mediating the phosphorylation of RIPK3 to promote endotoxin tolerance.

TRAF-associated NF-κB activator (TANK)-binding kinase 1 (TBK1), a nonclassical IκB kinase (IKK), and its effect on inflammation have not been entirely clarified. Here, we identified that TBK1 participates in the catabolism of glutamine by mediating the phosphorylation of receptor-interacting protein kinase 3 (RIPK3) and promoting macrophage endotoxin tolerance (ET). We found that the TBK1 protein directly interacts with the RIPK3 protein and mediates the phosphorylation of RIPK3 in macrophages. Activated RIPK3 can directly bind to glutamate dehydrogenase 1 (GLUD1), which is known to be a critical enzyme for catalyzing glutamine decomposition, to improve its catalytic activity and increase the production of α-ketoglutarate (α-KG) in macrophages. α-KG generated from glutaminolysis can promote M2 activation and restrict M1 polarization, which plays a crucial role in promoting lipopolysaccharide (LPS)-induced ET. As a result of TBK1 regulating the phosphorylation level of RIPK3, overexpressed TBK1 could enhance the tolerance of macrophages to endotoxin through glutaminolysis. Overall, these findings reveal a novel mechanism for the metabolic control of inflammation and for the induction of ET by modulating glutamine metabolism.

KDM6A Regulates Cell Plasticity and Pancreatic Cancer Progression by Noncanonical Activin Pathway.

BACKGROUND & AIMS: Inactivating mutations of KDM6A, a histone demethylase, were frequently found in pancreatic ductal adenocarcinoma (PDAC). We investigated the role of KDM6A (lysine demethylase 6A) in PDAC development. METHODS: We performed a pancreatic tissue microarray analysis of KDM6A protein levels. We used human PDAC cell lines for KDM6A knockout and knockdown experiments. We performed bromouridine sequencing analysis to elucidate the effects of KDM6A loss on global transcription. We performed studies with Ptf1aCre; LSL-KrasG12D; Trp53R172H/+; Kdm6afl/fl or fl/Y, Ptf1aCre; Kdm6afl/fl or fl/Y, and orthotopic xenograft mice to investigate the impacts of Kdm6a deficiency on pancreatic tumorigenesis and pancreatitis. RESULTS: Loss of KDM6A was associated with metastasis in PDAC patients. Bromouridine sequencing analysis showed up-regulation of the epithelial-mesenchymal transition pathway in PDAC cells deficient in KDM6A. Loss of KDM6A promoted mesenchymal morphology, migration, and invasion in PDAC cells in vitro. Mechanistically, activin A and subsequent p38 activation likely mediated the role of KDM6A loss. Inhibiting either activin A or p38 reversed the effect. Pancreas-specific Kdm6a-knockout mice pancreata showed accelerated PDAC progression, developed a more aggressive undifferentiated type of PDAC, and increased metastases in the background of Kras and p53 mutations. Kdm6a-deficient pancreata in a pancreatitis model had a delayed recovery with increased PDAC precursor lesions compared with wild-type pancreata. CONCLUSIONS: Loss of KDM6A accelerates PDAC progression and metastasis, most likely by a noncanonical p38-dependent activin A pathway. KDM6A also promotes pancreatic tissue recovery from pancreatitis. Activin A might be used as a therapeutic target for KDM6A-deficient PDACs.

Cyst fluid metabolites distinguish malignant from benign pancreatic cysts.

OBJECTIVES: Current standard of care imaging, cytology, or cystic fluid analysis cannot reliably differentiate malignant from benign pancreatic cystic neoplasms. This study sought to determine if the metabolic profile of cystic fluid could distinguish benign and malignant lesions, as well as mucinous and non-mucinous lesions. METHODS: Metabolic profiling by untargeted mass spectrometry and quantitative nuclear magnetic resonance was performed in 24 pancreatic cyst fluid from surgically resected samples with pathological diagnoses and clinicopathological correlation. RESULTS: (Iso)-butyrylcarnitine distinguished malignant from benign pancreatic cysts, with a diagnostic accuracy of 89%. (Iso)-butyrylcarnitine was 28-fold more abundant in malignant cyst fluid compared with benign cyst fluid (P=.048). Furthermore, 5-oxoproline (P=.01) differentiated mucinous from non-mucinous cysts with a diagnostic accuracy of 90%, better than glucose (82% accuracy), a previously described metabolite that distinguishes mucinous from non-mucinous cysts. Combined analysis of glucose and 5-oxoproline did not improve the diagnostic accuracy. In comparison, standard of care cyst fluid carcinoembryonic antigen (CEA) and cytology had a diagnostic accuracy of 40% and 60% respectively for mucinous cysts. (Iso)-butyrylcarnitine and 5-oxoproline correlated with cyst fluid CEA levels (P<.0001 and P<.05 respectively). For diagnosing malignant pancreatic cysts, the diagnostic accuracies of cyst size > 3 cm, ≥ 1 high-risk features, cyst fluid CEA, and cytology are 38%, 75%, 80%, and 75%, respectively. CONCLUSIONS: (Iso)-butyrylcarnitine has potential clinical application for accurately distinguishing malignant from benign pancreatic cysts, and 5-oxoproline for distinguishing mucinous from non-mucinous cysts.

Macrophage Membrane-Coated Nanoparticles Alleviate Hepatic Ischemia-Reperfusion Injury Caused by Orthotopic Liver Transplantation by Neutralizing Endotoxin.

PURPOSE: To investigate the effect and mechanism of macrophage membrane-coated nanoparticles (M-NPs) on hepatic ischemia-reperfusion injury (I/RI) caused by orthotopic liver transplantation. In addition, the advantages of TLR4+/M-NPs compared to M-NPs are discussed. MATERIALS AND METHODS: We prepared biomimetic M-NPs and identified their characteristics. M-NPs were injected into an SD rat model of orthotopic liver transplantation, and the anti-inflammatory and anti-I/RI activities of M-NPs were studied in vivo and in vitro. In addition, we overexpressed macrophage membrane Toll-like receptor 4 (TLR4) in vitro and prepared TLR4+/M-NPs. Then, we assessed the characteristics and advantages of TLR4+/M-NPs. RESULTS: The M-NPs neutralized endotoxin, inhibited the overactivation of Kupffer cells (KCs) and suppressed the secretion of inflammatory factors by inhibiting the endotoxin-mediated TLR4/MyD88/IRAK1/NF-κB signaling pathway. In an orthotopic liver transplantation model in SD rats, M-NPs showed significant therapeutic efficacy by neutralizing endotoxin and suppressing the secretion of inflammatory factors. Moreover, overexpression of TLR4 on the macrophage membrane by using a TLR4+-plasmid in vitro effectively reduced the amount of M-NPs needed to neutralize an equivalent dose of endotoxin, reducing the potential risks of NP overuse. CONCLUSION: This study indicates that M-NPs can effectively alleviate I/RI induced by liver transplantation.

STAT5a induces endotoxin tolerance by alleviating pyroptosis in kupffer cells.

Pyroptosis, a newly discovered type of programmed cell death, affects endotoxin tolerance in macrophages. However, the factors acting on the nod-like receptor 3 (Nlrp3) inflammasome and caspase1 activation to impede pyroptosis and resulting in tolerance and survival in sepsis were needed to discovered. Here, we found that signal transducer and activator of transcription 5A (STAT5a) restrains pyroptosis in Kupffer cells (KCs) and induces endotoxin tolerance (ET) in a sepsis model. The lentiviral knockdown of STAT5a led to enhanced pyroptosis in KCs, increased IL-1ß production and decreased IL-10 production via intricate NF-κb signaling regulation. Thus, our findings reveal a novel mechanism of STAT5a-midiated endotoxin tolerance in KCs.

Total Antioxidant Capacity and Pancreatic Cancer Incidence and Mortality in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial.

BACKGROUND: Total antioxidant capacity (TAC) reflects an individual's overall antioxidant intake. We sought to clarify whether higher TAC is associated with lower risks of pancreatic cancer incidence and mortality in the U.S. general population. METHODS: A total of 96,018 American adults were identified from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. A ferric-reducing ability of plasma score was used to reflect an individual's TAC intake from diet and/or supplements. Cox regression was used to calculate hazard ratios (HR) for pancreatic cancer incidence, and competing risk regression was used to calculate subdistribution HRs for pancreatic cancer mortality. Restricted cubic spline regression was used to test nonlinearity. RESULTS: A total of 393 pancreatic cancer cases and 353 pancreatic cancer-related deaths were documented. Total (diet + supplements) TAC was found to be inversely associated with pancreatic cancer incidence (HR quartile 4 vs. quartile 1 = 0.53; 95% confidence interval, 0.39-0.72; P trend = 0.0002) and mortality (subdistribution HR quartile 4 vs. quartile 1 = 0.52; 95% confidence interval 0.38-0.72; P trend = 0.0003) in a nonlinear dose-response manner (all P nonlinearity < 0.01). Similar results were observed for dietary TAC. No association of supplemental TAC with pancreatic cancer incidence and mortality was found. CONCLUSIONS: In the U.S. general population, dietary but not supplemental TAC level is inversely associated with risks of pancreatic cancer incidence and mortality in a nonlinear dose-response pattern. IMPACT: This is the first prospective study indicating that a diet rich in antioxidants may be beneficial in decreasing pancreatic cancer incidence and mortality.

Activator-Mediated Pyruvate Kinase M2 Activation Contributes to Endotoxin Tolerance by Promoting Mitochondrial Biogenesis.

Pyruvate kinase M2 (PKM2) is a key glycolysis enzyme, and its effect on macrophages has not been entirely elucidated. Here, we identified that the PKM2 small-molecule agonist TEPP-46 mediated PKM2 activation by inducing the formation of PKM2 tetramer and promoted macrophage endotoxin tolerance. Lipopolysaccharide (LPS)-tolerant mice had higher expression of the PKM2 tetramer, which was associated with a reduced in vivo immune response to LPS. Pretreatment of macrophages with TEPP-46 resulted in tolerance to LPS stimulation, as demonstrated by a significant reduction in the production of TNF-α and IL-6. We found that TEPP-46 induced mitochondrial biogenesis in macrophages. Inhibition of mitochondrial biogenesis by mtTFA knockdown effectively inhibited TEPP-46-mediated macrophage tolerance to endotoxins. We discovered that TEPP-46 promoted the expression of PGC-1α and that PGC-1α was the key regulator of mitochondrial biogenesis in macrophages induced by TEPP-46. PGC-1α was negatively regulated by the PI3K/Akt signaling pathway. Knockdown of PKM2 or PGC-1α uniformly inhibited TEPP-46-mediated endotoxin tolerance by inhibiting mitochondrial biogenesis. In addition, TEPP-46 protected mice from lethal endotoxemia and sepsis. Collectively, these findings reveal novel mechanisms for the metabolic control of inflammation and for the induction of endotoxin tolerance by promoting mitochondrial biogenesis. Targeting PKM2 appears to be a new therapeutic option for the treatment of sepsis and other inflammatory diseases.

Transcriptional co-regulator RIP140: An important mediator of the inflammatory response and its associated diseases (Review).

The inflammatory response is a physiological process that is essential for maintaining homeostasis of the immune system. Inflammation is classified into acute inflammation and chronic inflammation, both of which pose a risk to human health. However, specific regulatory mechanisms of the inflammatory response remain to be elucidated. Receptor interacting protein (RIP) 140 is a nuclear receptor that affects an extensive array of biological and pathological processes in the body, including energy metabolism, inflammation and tumorigenesis. RIP140­mediated macrophage polarization is important in regulating the inflammatory response. Overexpression of RIP140 in macrophages results in M1­like polarization and expansion during the inflammatory response. Conversely, decreased expression of RIP140 in macrophages reduces the number of M1­like macrophages and increases the number of alternatively polarized cells, which collectively promote endotoxin tolerance (ET) and relieve inflammation. This review summarizes the role of RIP140 in acute and chronic inflammatory diseases, with a focus on insulin resistance, atherosclerosis, sepsis and ET.

Overexpression of RIP140 suppresses the malignant potential of hepatocellular carcinoma by inhibiting NF­κB­mediated alternative polarization of macrophages.

Tumor-associated macrophages (TAMs) and their alternative activation contribute greatly to the development of hepatocellular carcinoma (HCC). Receptor-interacting protein 140 (RIP140) is widely expressed in macrophages and regulates macrophage-mediated energy metabolism, the inflammatory response and tumorigenesis. However, whether RIP140 is involved in the activation of TAMs has not been reported. In the present study, we determined the expression of RIP140 in macrophages after treatment with HCC-conditioned medium (HCM) for 24 h. We also analyzed the effects of RIP140 overexpression on macrophage polarization, invasion and apoptosis of HepG2 and Huh7 cells. Transwell and apoptosis assays were used to estimated cell invasion and apoptosis. In addition, we investigated the effects of RIP140 overexpression in macrophages on the growth of H22 cells by subcutaneous injection of H22 cells along with macrophages in BALB/c nude mice. Western blotting and qRT-PCR were used to detect protein and mRNA expression associated with the NF-κB/IL-6 axis in TAMs. Immunohistochemical and immunofluorescence staining were used to evaluate the protein expression of RIP140 or F4/80 in human HCC samples. The protein expression of RIP140 in peripheral blood mononuclear cells was detected by western blotting. Kaplan­Meier survival curve estimation of overall survival for patients with HCC was based on RIP140 or F4/80 expression in HCC samples. We found that HCM inhibited RIP140 expression and fostered the alternative activation of macrophages. RIP140 overexpression in TAMs significantly inhibited the alternative activation of macrophages by inhibiting the NF-κB/IL-6 axis in TAMs, and suppressed HCC cell growth both in vitro and in vivo. In addition, the protein expression of RIP140 in peripheral blood monocytes was significantly lower in patients with HCC than in healthy people, and this result was consistent with the expression of RIP140 in HCC samples. Furthermore, low RIP140 expression and high F4/80 expression were found to be closely correlated with shorter survival time of the patients with HCC.

[Over-expression of receptor-interacting protein 140 in tumor-associated macrophages suppresses invasion and proliferation of hepatoma cells in vitro].

OBJECTIVE: To investigate the role of receptor-interacting protein 140 (RIP140) in tumor-associated macrophages (TAMs) in the invasion and proliferation of hepatoma cells in vitro. METHODS: Western blotting, qRT-PCR and flow cytometry were performed to examine the effects of lentivirus-mediated RIP140 over-expression in mouse peritoneal macrophages (PMs). Western blotting, qRT-PCR and immunofluorescence staining were used to detect the expression of RIP140 in TAMs following stimulation of the PMs with hepatocellular carcinoma conditioned medium (HCM) for 24 h. The polarization index and the expression of NF-κB and IL-6 were detected using qRT-PCR in TAMs in HCM-stimulated PMs with or without RIP140 over-expression. Transwell assay and flow cytometry were used to estimate the cell invasion and apoptosis. HE staining and immunohistochemical staining were used to analyze the effects of RIP140-over-expressing macrophages on the growth and tumor formation of H22 cells in BALB/c nude mice. RESULTS: The lentivirus vector efficiently mediated RIP140 over-expression in mouse PMs. HCM stimulation significantly inhibited RIP140 expression in the TAMs and promoted their M2-like polarization. Over-expression of RIP140 in PMs suppressed the invasion and induced apoptosis of HCC cells. RIP140 over-expression inhibited HCM-induced M2 polarization and the activation of NF-κB/IL-6 axis in the TAMs, and RIP140- overexpressing TAMs obviously suppressed the growth of H22 cell xenograft in nude mice. CONCLUSION: Over-expression of RIP140 in TAMs suppresses the growth and proliferation of hepatoma cells possibly by inhibiting M2 polarization of the TAMs.