Dexmedetomidine attenuates sepsis-associated acute lung injury by regulating macrophage efferocytosis through the ROS/ADAM10/AXL pathway.

BACKGROUND: The lungs are highly susceptible to damage during sepsis, with severe lung injury potentially progressing to acute respiratory distress syndrome and even fatal sepsis. Effective efferocytosis of apoptotic cells is crucial in alleviating inflammation and tissue injury. METHODS: We established a septic lung injury mouse model via intraperitoneal injection of lipopolysaccharide. Lung injury was assessed by histology, immunofluorescence, neutrophil immunohistochemistry staining, and cytokine detection. We extracted alveolar macrophages by bronchoalveolar lavage and primary macrophages from mouse bone marrow to investigate the regulatory effects of Dexmedetomidine (DEX) on efferocytosis. We further validated the molecular mechanisms underlying the regulation of macrophage efferocytosis by DEX through knockdown of AXL expression. Additionally, we examined the efferocytic ability of monocytes isolated from patients. RESULTS: We discovered that DEX treatment effectively alleviated pulmonary injury and inflammation. Lipopolysaccharide reduced macrophage efferocytosis and AXL expression which were reversed by DEX. We also found DEX inhibited the increased activation of A Disintegrin And Metalloproteinase 10 (ADAM10) and the production of soluble AXL. Moreover, our findings demonstrated that DEX decreased the elevated ROS production linked to higher ADAM10 activation. Blocking AXL negated DEX's benefits on efferocytosis and lung protection. Efferocytosis in monocytes from septic lung injury patients was notably lower than in healthy individuals. CONCLUSION: Our findings demonstrated that DEX treatment effectively reduces septic lung injury by promoting macrophage efferocytosis through ROS/ADAM10/AXL signaling pathwway.

Macrophage STING signaling promotes NK cell to suppress colorectal cancer liver metastasis via 4-1BBL/4-1BB co-stimulation.

BACKGROUND AND AIMS: Macrophage innate immune response plays an important role in tumorigenesis. However, the role and mechanism of macrophage STING signaling in modulating tumor microenvironment to suppress tumor growth at secondary sites remains largely unclear. METHODS: STING expression was assessed in liver samples from patients with colorectal cancer (CRC) liver metastasis. Global or myeloid stimulator of interferon gene (STING)-deficient mice, myeloid NOD-like receptor protein 3 (NLRP3)-deficient mice, and wild-type (WT) mice were subjected to a mouse model of CRC liver metastasis by intrasplenic injection of murine colon carcinoma cells (MC38). Liver non-parenchymal cells including macrophages and natural killer (NK) cells were isolated for flow cytometry analysis. Bone marrow-derived macrophages pretreated with MC38 were co-cultured with splenic NK cells for in vitro studies. RESULTS: Significant activation of STING signaling were detected in adjacent and tumor tissues and intrahepatic macrophages. Global or myeloid STING-deficient mice had exacerbated CRC liver metastasis and shorten survival, with decreased intrahepatic infiltration and impaired antitumor function of NK cells. Depletion of NK cells in WT animals increased their metastatic burden, while no significant effects were observed in myeloid STING-deficient mice. STING activation contributed to the secretion of interleukin (IL)-18 and IL-1ß by macrophages, which optimized antitumor activity of NK cells by promoting the expression of 4-1BBL in macrophages and 4-1BB in NK cells, respectively. Moreover, MC38 treatment activated macrophage NLRP3 signaling, which was inhibited by STING depletion. Myeloid NLRP3 deficiency increased tumor burden and suppressed activation of NK cells. NLRP3 activation by its agonist effectively suppressed CRC liver metastasis in myeloid SITNG-deficient mice. CONCLUSIONS: We demonstrated that STING signaling promoted NLRP3-mediated IL-18 and IL-1ß production of macrophages to optimize the antitumor function of NK cells via the co-stimulation signaling of 4-1BBL/4-1BB.

Defective mitophagy in aged macrophages promotes mitochondrial DNA cytosolic leakage to activate STING signaling during liver sterile inflammation.

Macrophage-stimulator of interferon genes (STING) signaling mediated sterile inflammation has been implicated in various age-related diseases. However, whether and how macrophage mitochondrial DNA (mtDNA) regulates STING signaling in aged macrophages remains largely unknown. We found that hypoxia-reoxygenation (HR) induced STING activation in macrophages by triggering the release of macrophage mtDNA into the cytosol. Aging promoted the cytosolic leakage of macrophage mtDNA and enhanced STING activation, which was abrogated upon mtDNA depletion or cyclic GMP-AMP Synthase (cGAS) inhibition. Aged macrophages exhibited increased mitochondrial injury with impaired mitophagy. Mechanistically, a decline in the PTEN-induced kinase 1 (PINK1)/Parkin-mediated polyubiquitination of mitochondria was observed in aged macrophages. Pink1 overexpression reversed the inhibition of mitochondrial ubiquitination but failed to promote mitolysosome formation in the aged macrophages. Meanwhile, aging impaired lysosomal biogenesis and function in macrophages by modulating the mTOR/transcription factor EB (TFEB) signaling pathway, which could be reversed by Torin-1 treatment. Consequently, Pink1 overexpression in combination with Torin-1 treatment restored mitophagic flux and inhibited mtDNA/cGAS/STING activation in aged macrophages. Moreover, besides HR-induced metabolic stress, other types of oxidative and hepatotoxic stresses inhibited mitophagy and promoted the cytosolic release of mtDNA to activate STING signaling in aged macrophages. STING deficiency protected aged mice against diverse types of sterile inflammatory liver injuries. Our findings suggest that aging impairs mitophagic flux to facilitate the leakage of macrophage mtDNA into the cytosol and promotes STING activation, and thereby provides a novel potential therapeutic target for sterile inflammatory liver injury in aged patients.

MGP promotes CD8+ T cell exhaustion by activating the NF-κB pathway leading to liver metastasis of colorectal cancer.

Matrix Gla protein (MGP) was originally reported as a physiological suppressor of ectopia calcification and has also been reported to be associated with cancer. However, the relation between the biological functions of MGP and the immune response in colorectal cancer (CRC) remains unclear. Here, we investigated the regulatory role of MGP in the immune microenvironment of CRC. MGP expression in CRC samples was assessed by single-cell RNA sequencing and the Gene Expression Omnibus (GEO) database, and confirmed by quantitative real-time Polymerase Chain Reaction (qRT-PCR) and immunohistochemistry analysis of human CRC samples. The effect of MGP on proliferation and invasion of CRC cells was evaluated by in vitro assays involving MGP knockdown and overexpression. Luciferase reporter assay and chromatin immunoprecipitation (ChIP)-qPCR assay were performed to identify transcriptional regulatory sites of the nuclear factor kappa-B (NF-κB) and programmed cell death ligand 1 (PD-L1). In vivo experiments were performed in mouse model of CRC liver metastasis established via spleen injection. The results revealed that MGP was significantly upregulated in cancer cell clusters from the primary CRC or liver metastases, compared with that in the corresponding paracancerous tissues via single-cell RNA sequencing. MGP enriched intracellular free Ca2+ levels and promoted NF-κB phosphorylation, thereby activated PD-L1 expression to promote CD8+ T cell exhaustion in CRC. The luciferase reporter assay and ChIP-qPCR assay indicated that the transcriptional regulation of NF-κB upregulated PD-L1 expression. In vivo, MGP inhibition significantly decreased the rate of CRC liver metastasis, which was further reduced after combined therapy with αPD1 (anti-PD1). In conclusions, this study revealed that MGP can facilitate CD8+ T cell exhaustion by activating the NF-κB pathway, leading to liver metastasis of CRC. The combination of MGP knockdown and αPD1 can synergistically resist liver metastasis of CRC.

m6A modification of circHPS5 and hepatocellular carcinoma progression through HMGA2 expression.

N6-methyladenosine (m6A) is capable of mediating circRNA generation in carcinoma biology. Nevertheless, the posttranscriptional systems of m6A and circRNA in hepatocellular carcinoma (HCC) development are still unclear. The present study identified a circRNA with m6A modification, circHPS5, which was increased in neoplasm HCC tissues and indicated poor patient survival. Silencing of circHPS5 inhibited epithelial-mesenchymal transition (EMT) and cancer stem-like cell (CSC) phenotypes. Notably, METTL3 could direct the formation of circHPS5, and specific m6A controlled the accumulation of circHPS5. YTHDC1 facilitated the cytoplasmic output of circHPS5 under m6A modification. In addition, we demonstrated that circHPS5 can act as a miR-370 sponge to regulate the expression of HMGA2 and further accelerate HCC cell tumorigenesis. Accordingly, the m6A modification of circHPS5 was found to modulate cytoplasmic output and increase HMGA2 expression to facilitate HCC development. The new regulatory model of "circHPS5-HMGA2" provides a new perspective for circHPS5 as an important prognostic marker and therapeutic target in HCC and provides mechanistic insight for exploring the carcinogenic mechanism of circHPS5 in HCC.

Dexmedetomidine preconditioning alleviated murine liver ischemia and reperfusion injury by promoting macrophage M2 activation via PPARγ/STAT3 signaling.

BACKGROUND: Although a protective role of dexmedetomidine in liver ischemia and reperfusion (IR) injury has been reported, the underlying mechanism remains to be determined. The aim of this study is to analyze the effects of dexmedetomidine on the regulation of macrophage innate immune activation during liver IR. METHODS: Mice were randomly divided into dexmedetomidine preconditioning (DEX) and phosphate buffered saline vehicle control (VEH) groups. A murine 70% warm liver IR model was used, and liver injury and intrahepatic inflammation was compared between groups. Bone marrow-derived macrophages (BMDMs) were stimulated with LPS in the presence or absence of dexmedetomidine. The inflammatory cytokine production was measured, and the macrophage M1/M2 polarization was determined in different groups. The underlying mechanism of dexmedetomidine in regulating macrophage M2 activation was also analyzed. RESULTS: Compared to mice observed in the control group, mice in the DEX group showed reduced liver injury and diminished proinflammatory immune responses in livers post IR. In vitro, dexmedetomidine pretreatment promoted BMDMs M2 activation, as evidenced by increased Arg1 and Mrc1 gene induction, decreased iNOS gene induction, inhibited phosphorated-signal transducer and activator of transcription 1 (p-STAT1) but enhanced p-STAT6 expression, much lower levels of proinflammatory TNF-α and IL-6, and higher levels of anti-inflammatory IL-10 cytokine secretion. Signaling pathway analysis revealed that peroxisome proliferator-activated receptor-γ (PPARγ)/ STAT3 activation was upregulated in BMDMs with dexmedetomidine pretreatment. Furthermore, PPARγ knockdown by siRNA not only inhibited STAT3 activation but also abrogated the promotion effects of macrophage M2 activation in BMDMs pretreated with dexmedetomidine. Finally, in vivo PPARγ inhibition in macrophages by siRNA significantly increased liver IR injury and intrahepatic inflammation in mice from the Dex group, with no significant effect in the VEH group. CONCLUSIONS: Our results indicate that dexmedetomidine preconditioning inhibited intrahepatic proinflammatory innate immune activation by promoting macrophage M2 activation in a PPARγ/STAT3 dependent manner. Our results demonstrate a novel innate immune regulatory mechanism by dexmedetomidine preconditioning during liver IR injury.

Obstructive jaundice secondary to fungal infection: a rare case report.

Obstructive jaundice is characterized by an obstruction of the intrahepatic or extrahepatic biliary system, and the most common causes include pancreatic and duodenal periampullary cancer. There have been some cases reporting obstructive jaundice caused by infection. Deep tissue infection usually develops in the individuals who are immunologically compromised or chronically ill, while a few cases reported in the immunocompetent patients. Those cases were diagnosed by fungal culture or percutaneous biopsy. Here, we presented an interesting case of obstructive jaundice secondary to fungal infection confirmed by postoperative pathological examination. A 79 years old man complaint about upper abdominal discomfort, darkened urine, and skin itch, with a history of esophageal cancer operation 5 years ago. The serology for hepatitis virus and human immunodeficiency virus (HIV) was negative. Imaging examinations showed a nodular located at distal common bile duct. As evidenced by increased level of cancer antigen 19-9 (CA19-9), the patient was highly suspected to be malignant obstructive jaundice. Thus, pylorus preserving pancreaticoduodenectomy (PPPD) was conducted. To our surprise, the ultimate diagnosis was fungal infection at the site of duodenum ampulla by the postoperative pathological examination, with no evidence of malignance. Anti-infective therapy was conducted subsequently, combined by fluconazole, sulperazone and tinidazole. Three weeks later, the patient was generally in good condition and discharged from hospital. During the 2-year follow-up, no fungal infection or tumor recurrence was observed. This case reminded us that fungal infection could be the cause of obstructive jaundice in an elderly person.

Aging aggravated liver ischemia and reperfusion injury by promoting hepatocyte necroptosis in an endoplasmic reticulum stress-dependent manner.

BACKGROUND: Aggravated liver ischemia and reperfusion (IR) injury has been reported in aged mice. Although necroptosis inhibition showed no crucial effect on hepatic IR injury in young mice, whether and how necroptosis affects liver IR injury in aged mice remains unclear. METHODS: Young and aged mice were subjected to liver IR modeling. Liver injury, hepatocyte necroptosis and endoplasmic reticulum (ER) stress were analyzed in different groups. RESULTS: Significantly increased liver necroptosis was found in aged mice post IR compared with young mice. Necroptosis inhibition by necrostatin-1 (Nec-1) decreased hepatocyte necroptosis and liver injury post IR in aged mice, with no significant effects on young mice. Furthermore, IR induced ER stress in both young and aged mice, and enhanced ER stress was observed in aged mice post IR. Administration of 4-phenylbutyrate (4-PBA), an ER stress antagonist, alleviated liver IR injury in both young and aged mice. However, ER stress inhibition reduced hepatocyte necroptosis in aged mice but not in young mice. CONCLUSIONS: Aging increased ER stress in IR-stressed hepatocytes, leading to aggravated necroptosis and liver IR injury. Our study demonstrated a novel mechanism of ER stress in the regulation of hepatocyte necroptosis in aged livers post IR, which would be a potential therapeutic target to reduce liver IR injury in elderly patients.

Aging aggravated liver ischemia and reperfusion injury by promoting STING-mediated NLRP3 activation in macrophages.

Although aggravated liver injury has been reported in aged livers post-ischemia and reperfusion (IR), the underlying mechanism of innate immune activation of aged macrophages is not well understood. Here, we investigated whether and how Stimulator of interferon genes (STING) signaling regulated macrophage proinflammatory activation and liver IR injury. Mice were subjected to hepatic IR in vivo. Macrophages isolated from IR-stressed livers and bone marrow-derived macrophages (BMDMs) from young and aged mice were used for in vitro studies. Enhanced nucleotide-binding domain and leucine-rich repeat containing protein 3 (NLRP3) activation was found in both livers and macrophages of aged mice post-IR. NLRP3 knockdown in macrophages inhibited intrahepatic inflammation and liver injury in both young and aged mice. Interestingly, enhanced activation of the STING/ TANK-binding kinase 1 (TBK1) signaling pathway was observed in aged macrophages post-IR and mitochondria DNA (mtDNA) stimulation. STING suppression blocked over-activation of NLRP3 signaling and excessive secretion of proinflammatory cytokines/chemokines in the mtDNA-stimulated BMDMs from aged mice. More importantly, STING knockdown in macrophages abrogated the detrimental role of aging in aggravating liver IR injury and intrahepatic inflammation. Finally, peripheral blood from the recipients undergoing liver transplantation was collected and analyzed. The results showed that the elderly recipients had much higher levels of TNF-α, IL-6, IL-1ß, and IL-18 post-transplantation, indicating increased NLRP3 activation in lR-stressed livers of elderly recipients. In summary, our study demonstrated that the STING-NLRP3 axis was critical for the proinflammatory response of aged macrophages and would be a novel therapeutic target to reduce IR injury in elderly patients.

N-acetylcysteine alleviates liver injury by suppressing macrophage-mediated inflammatory response post microwave ablation.

OBJECT: To investigate N-acetyl-cysteine (NAC) would able to alleviate liver injury and systemic inflammatory response caused by microwave ablation (MWA) in rats. MATERIALS AND METHODS: Male Sprague-Dawley rats weighing 150-200 g were randomly divided into sham group (only anesthesia and laparotomy except MWA but with intraperitoneal PBS or NAC solution injection according to different situations), control group (intraperitoneal PBS injection for comparation 2 h prior to MWA), and NAC-treated group (intraperitoneal N-acetyl-cysteine (300 mg/kg) injection 2 h prior to MWA). Experimental rats were sacrificed at 4 h following operation in line with the liver injury severity curve. Liver tissue and serum samples were collected for determination of pathology, apoptosis, macrophages contents and protein expression. RESULTS: The elevated serum level of liver enzymes, Myeloperoxidase (MPO) and inflammatory factors (TNF-α and CXCL1) in MWA-treated rats revealed injurious and pro- inflammatory effect of MVA. Macrophages aggregation was detected in MWA exposure rats similarly. and NAC pre-conditioning mitigate liver damage and hepatocyte apoptosis, besides macrophages accumulation and following inflammatory response in liver tissue. CONCLUSION: Our results demonstrated that N-acetyl-cysteine application alleviate macrophages aggregation and inflammatory response in liver suffering microwave ablation, and mitigating liver injury and cell apoptosis.

Combined ischemic and rapamycin preconditioning alleviated liver ischemia and reperfusion injury by restoring autophagy in aged mice.

Old livers are more damaged by hepatic ischemia and reperfusion (IR) injury than young livers. The aim of this study was to investigate the effects of ischemic and rapamycin preconditioning on IR injury in old livers. Young (8-week-old) and aged (60-week-old) mice were subjected to IR or a sham control procedure. The aged mice were randomly divided into six groups: IR (CON), IR with ischemic preconditioning (IPC), IR with rapamycin preconditioning (RAPA), IR with combined ischemic and rapamycin preconditioning (IPC + RAPA), IR with 3-methyladenine (3-MA), IR with combined ischemic and rapamycin preconditioning with 3-MA pretreatment (IPC + RAPA+3-MA). Liver injury was evaluated 6 h after reperfusion. Hepatocellular autophagy induction was also analyzed by western blotting. The results revealed that aged mice had aggravated liver IR injury as compared to young mice. In aged mice following IR, IPC + RAPA but not IPC or RAPA alleviated liver injury, as evidenced by lower levels of serum ALT, improved preservation of liver architecture with lower Suzuki scores, and decreased caspase-3 activity compared with CON. In addition, western blot analysis revealed increased LC3B II but decreased p62 protein expression levels in the IPC + RAPA group, indicating that autophagic flux was restored by combined ischemic and rapamycin preconditioning. Furthermore, autophagy inhibition by the inhibitor 3-MA abrogated the protective role in the IPC + RAPA group, while no significant effects were observed in the CON group. In conclusions, our results demonstrated that combined ischemic and rapamycin preconditioning protected old livers against IR injury, which was likely attributed to restored autophagy activation.