Oroxylin A-induced Trained Immunity Promotes LC3-associated Phagocytosis in Macrophage in Protecting Mice Against Sepsis.

Sepsis is defined as a dysregulated host response to infection that leads to multiorgan failure. Innate immune memory, i.e., "trained immunity", can result in stronger immune responses and provide protection against various infections. Many biological agents, including ß-glucan, can induce trained immunity, but these stimuli may cause uncontrolled inflammation. Oroxylin A (OA) is an active flavonoid compound that is derived from Scutellaria baicalensis. OA is an agonist for inducing trained immunity in vivo and in vitro, and ß-glucan was used as a positive control. The protective effects of OA-induced trained immunity were evaluated in mouse models that were established by either lipopolysaccharide (LPS) administration or caecal ligation and puncture (CLP). The expression of inflammatory factors and signaling pathway components involved in trained immunity was evaluated in vitro using qRT‒PCR, western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). Flow cytometry and confocal microscopy were used to examine reactive oxygen species (ROS) levels and phagocytosis in trained macrophages. A PCR array was used to screen genes that were differentially expressed in trained macrophages. Here, we revealed that OA alleviated sepsis via trained immunity. OA-treated macrophages displayed increased glycolysis and mTOR phosphorylation, and mTOR inhibitors suppressed OA-induced trained immunity by effectively reprogramming macrophages. The PCR array revealed key genes in the mTOR signaling pathway in OA-treated macrophages. Furthermore, OA targeted the Dectin-1-syk axis to promote LC3-associated phagocytosis (LAP) by trained macrophages, thereby enhancing the ability of these macrophages to protect against infection. This ability could be transferred to a new host via the adoptive transfer of peritoneal macrophages. This study is the first to provide new insights into the potential of OA-induced trained immunity to be used as a strategy to protect mice against sepsis by promoting LAP by macrophages.

2bRAD-M reveals the difference in microbial distribution between cancerous and benign ovarian tissues.

The development of ovarian cancer is closely related to various factors, such as environmental, genetic and microbiological factors. In previous research, bacteria were identified in human tumors by 16S rRNA sequencing. However, the microbial biomass in tumor tissue is too low and cannot be accurately identified by 16S rRNA sequencing. In our study, we employ 2bRAD sequencing for Microbiome (2bRAD-M), a new sequencing technology capable of accurately characterizing the low biomass microbiome (bacteria, fungi and archaea) at species resolution. Here we surveyed 20 ovarian samples, including 10 ovarian cancer samples and 10 benign ovarian samples. The sequencing results showed that a total of 373 microbial species were identified in both two groups, of which 90 species shared in the two groups. The Meta statistic indicated that Chlamydophila_abortus and CAG-873_sp900550395 were increased in the ovarian cancer tissues, while Lawsonella_clevelandensis_A, Ralstonia_sp001078575, Brevundimonas_aurantiaca, Ralstonia_sp900115545, Ralstonia_pickettii, Corynebacterium_kefirresidentii, Corynebacterium_sp000478175, Brevibacillus_D_fluminis, Ralstonia_sp000620465, and Ralstonia_mannitolilytica were more abundant in the benign ovarian tissues. This is the first use of 2bRAD-M technique to provide an important hint for better understanding of the ovarian cancer microbiome.

Quxie Capsule Alleviates Colitis-associated Colorectal Cancer Through Modulating the Gut Microbiota and Suppressing A. fumigatus-induced Aerobic Glycolysis.

AIM: Quxie capsule (QX), a compound of 21 kinds of Traditional Chinese Medicine (TCM) herbs, has been used to treat patients with metastatic colorectal cancer (mCRC) and could suppress the growth of colon cancer. However, the mechanisms of QX inhibiting colorectal cancer remain unclear. In current study, we attempted to determine the anti-colorectal cancer (CRC) effects of QX and the mechanisms of QX in alleviating colorectal cancer. METHODS: A colitis-associated colon cancer (CAC) model was established by intraperitoneally injecting mice with AOM followed by 3 cycles of 2% DSS in water. During establishment of CAC model, we orally gavaged mice with QX. Hematoxylin and eosin (H&E) and immunohistochemistry were performed to assess lesion of the colonic tumors. The expression of pro-inflammatory cytokines in colonic tumors was measured by qPCR. The proportion of immune cells in colonic tumors was analyzed by flow cytometry. Internal transcribed spacer (ITS) sequencing and 16S rRNA gene sequencing were performed to detect intestinal microbiota. The expression of glycolytic related enzymes, lactate production, and extracellular acidification rate (ECAR) were used to assess the level of aerobic glycolysis. RESULTS: QX markedly inhibited intestinal tumorigenesis by decreasing the expression of pro-inflammatory cytokines and the proportion of myeloid-derived suppressor cells (MDSCs), and increasing the proportion of CD8+ T cells in colon tumors. Fecal microbiota sequencing revealed that QX increased the relative abundances of intestinal symbiotic probiotics, such as, Lactobacillus, Bifidobacterium and Faecalibacterium genera. What's more, opportunistic pathogens, Bacteroides genera and Aspergillus-Aspergillus fumigatus, exhibited remarkably reduced abundances in mice treated with QX compared with untreated CAC mice. Further experiments showed that QX significantly reduced glycolysis of colon tumor and suppressed A. fumigatus-induced glycolytic metabolism of colon cancer cells. CONCLUSIONS: QX alleviates the development of CRC at least in part through modulating intestinal microbiota and reducing A. fumigatus-induced aerobic glycolysis of colon cancer cells.

Candida tropicalis induces NLRP3 inflammasome activation via glycogen metabolism-dependent glycolysis and JAK-STAT1 signaling pathway in myeloid-derived suppressor cells to promote colorectal carcinogenesis.

Our previous studies showed that Candida tropicalis promoted colorectal cancer (CRC) by activating the function of MDSCs. However, underlying molecular mechanisms remains to be further investigated. In the present study, we indicated that C. tropicalis induced NLRP3 inflammasome activation through Dectin-3 in myeloid-derived suppressor cells (MDSCs). Mechanistically, we identified that C. tropicalis significantly enhanced the levels of glycolysis dependent on glycogen metabolism in MDSCs, which was required for NLRP3 inflammasome activation. C. tropicalis-induced NLRP3 inflammasome activation of MDSCs required the first priming signal and the second activation signal. For one thing, C. tropicalis promoted transcription of Nlrp3, Pro-caspase-1 and IL-1ß genes through activation of JAK-STAT1 signaling pathway. For another, mtROS as the second activation signal mediated C. tropicalis-induced activation of NLRP3 inflammasome. Pharmacological inhibition of NLRP3 inflammasome activation abolished the pro-tumorigenic effect of C. tropicalis in an AOM/DSS-induced CAC mice model and significantly reduced C. tropicalis-promoted infiltration of MDSCs in colon tumors. Finally, in human CRC samples, the expression of STAT1, p-STAT1 and NLRP3 was elevated in MDSCs infiltrated by CRC. Collectively, these findings shed light on a previously unidentified mechanism by which C. tropicalis induces NLRP3 inflammasome activation in MDSCs to contribute to the progression of CRC. And STAT1-NLRP3 axis might represent a prospective therapeutic target for the treatment of CRC.

Gut fungi enhances immunosuppressive function of myeloid-derived suppressor cells by activating PKM2-dependent glycolysis to promote colorectal tumorigenesis.

BACKGROUND: Accumulating evidence implicates that gut fungi are associated with the pathogenesis of colorectal cancer (CRC). Our previous study has revealed that Candida tropicalis (C. tropicalis) promotes colorectal tumorigenesis by enhancing immunosuppressive function of myeloid-derived suppressor cells (MDSCs) and increasing accumulation of MDSCs, but the underlying mechanisms remain unestablished. METHODS: Bone marrow-derived MDSCs were stimulated with C. tropicalis. RNA-sequencing analysis was performed to screen the differentially expressed genes. Quantitative real-time PCR and western blot were used to measure the expression of related proteins. Co-culture assay of MDSCs and CD8+ T cells was used to determine the immunosuppressive ability of MDSCs. Metabolomic analysis was conducted to detect metabolic reprogramming of MDSCs. Aerobic glycolysis of MDSCs was assessed by extracellular acidification rate (ECAR), glucose consumption and lactate production. A CAC mouse model was induced by AOM and DSS to determine the therapeutic action of TEPP-46. IHC and immunofluorescence were performed to examine the expression of PKM2, PKM2 (p-Y105) and iNOS in human CRC-infiltrated MDSCs. RESULTS: C. tropicalis facilitates immunosuppressive function of MDSCs by increasing the expression of iNOS, COX2 and NOX2, production of nitric oxide (NO) and reactive oxygen species (ROS). Mechanistically, C. tropicalis facilitates the immunosuppressive function of MDSCs through the C-type lectin receptors Dectin-3 and Syk. C. tropicalis-enhanced immunosuppressive function of MDSCs is further dependent on aerobic glycolysis. On the one hand, NO produced by MDSCs enhanced aerobic glycolysis in a positive feedback manner. On the other hand, C. tropicalis promotes p-Syk binding to PKM2, which results in PKM2 Tyr105 phosphorylation and PKM2 nuclear translocation in MDSCs. Nuclear PKM2 interacts with HIF-1α and subsequently upregulates the expression of HIF-1α target genes encoding glycolytic enzymes, GLUT1, HK2, PKM2, LDHA and PDK1, which are required for the C. tropicalis-induced aerobic glycolysis of MDSCs. Blockade of PKM2 nuclear translocation attenuates C. tropicalis-mediated colorectal tumorigenesis. The high expression of PKM2, PKM2 (p-Y105) and iNOS in CRC-infiltrated MDSCs correlates with the development of human CRC. CONCLUSION: C. tropicalis enhances immunosuppressive function of MDSCs via Syk-PKM2-HIF-1α-glycolysis signaling axis, which drives CRC. Therefore, we identify the Syk-PKM2-HIF-1α-glycolysis signaling axis as a potential therapeutic target for CRC.