CD11c+ myeloid cell exosomes reduce intestinal inflammation during colitis.

Intercellular communication is critical for homeostasis in mammalian systems, including the gastrointestinal (GI) tract. Exosomes are nanoscale lipid extracellular vesicles that mediate communication between many cell types. Notably, the roles of immune cell exosomes in regulating GI homeostasis and inflammation are largely uncharacterized. By generating mouse strains deficient in cell-specific exosome production, we demonstrate deletion of the small GTPase Rab27A in CD11c+ cells exacerbated murine colitis, which was reversible through administration of DC-derived exosomes. Profiling RNAs within colon exosomes revealed a distinct subset of miRNAs carried by colon- and DC-derived exosomes. Among antiinflammatory exosomal miRNAs, miR-146a was transferred from gut immune cells to myeloid and T cells through a Rab27-dependent mechanism, targeting Traf6, IRAK-1, and NLRP3 in macrophages. Further, we have identified a potentially novel mode of exosome-mediated DC and macrophage crosstalk that is capable of skewing gut macrophages toward an antiinflammatory phenotype. Assessing clinical samples, RAB27A, select miRNAs, and RNA-binding proteins that load exosomal miRNAs were dysregulated in ulcerative colitis patient samples, consistent with our preclinical mouse model findings. Together, our work reveals an exosome-mediated regulatory mechanism underlying gut inflammation and paves the way for potential use of miRNA-containing exosomes as a novel therapeutic for inflammatory bowel disease.

Relevance of miR-223 as Potential Diagnostic and Prognostic Markers in Cancer.

In 1993, the discovery of microRNAs in Caenorhabditis elegans (C. elegans) altered the paradigmatic view of RNA biology and post-transcriptional gene regulation. Further study revealed the role of microRNAs in disease development and progression. In particular, this review highlights microRNA-223 (miR-223 or miRNA-223) expression in malignant neoplastic disorders. miR-223 expression controls aspects of hematopoiesis and apoptosis, and cell proliferation, migration, and invasion. miR-223 regulates a number of gene targets, including cytoplasmic activation/proliferation-associated protein-1 (Caprin-1), insulin-like growth factor-1 receptor (IGF-1R), and other cell proliferation- and cell cycle-associated genes. Several studies have proposed miR-223 as a novel biomarker for early cancer diagnosis. Here, we emphasize miR-223's role in the development and progression of cancer.

Gastric tumorigenesis induced by combining Helicobacter pylori infection and chronic alcohol through IL-10 inhibition.

Helicobacter pylori infection and alcohol intake are independent risk factors in gastric carcinogenesis; however, until now, the combined effect of H. pylori infection and alcohol consumption and the specific mechanism is still problematic. Here, we developed a series of mouse models that progress from chronic gastritis to gastric cancer, induced by infecting H. pylori combined with chronic alcohol consumption and then determining the molecular mechanism of the progression by flow cytometry, western blotting, qPCR, Mito Traker assay in the gastric cancer and T-cell lines. Interleukin-10 (IL-10) knockout mice was used to determine whether IL-10 deficiency directly contributes to H. pylori and alcohol induced gastric tumorigenesis. Alcohol consumption, together with H. pylori infection, causes gastric cancer; IL-10 downregulation and mitochondrial metabolic dysfunction in CD8+ cells are also involved. IL-10 knockout accelerates tumor development in mice with either H. pylori infection or alcohol induced gastric cancer or both. IL-10 inhibits glucose uptake and glycolysis and promotes oxidative phosphorylation with lactate inhibition. Consequently, in the absence of IL-10 signaling, CD8+ cells accumulate damaged mitochondria in a mouse model of gastric cancer induced with the combination of alcohol plus H. pylori infection, and this results in mitochondrial dysfunction and production of IL-1ß. IL-1ß promotes H. pylori infection and reduces NKX6.3 gene expression, resulting in increased cancer cell survival and proliferation. Gastric cancer can be induced by the combination of H. pylori infection and chronic alcohol consumption through IL-10 inhibition induced CD8+ cells dysfunction and NKX6.3 suppression.

Epithelial-myeloid exchange of MHC class II constrains immunity and microbiota composition.

Intestinal epithelial cells (IECs) have long been understood to express high levels of major histocompatibility complex class II (MHC class II) molecules but are not considered canonical antigen-presenting cells, and the impact of IEC-MHC class II signaling on gut homeostasis remains enigmatic. As IECs serve as the primary barrier between underlying host immune cells, we reasoned that IEC-intrinsic antigen presentation may play a role in responses toward the microbiota. Mice with an IEC-intrinsic deletion of MHC class II (IECΔMHC class II) are healthy but have fewer microbial-bound IgA, regulatory T cells (Tregs), and immune repertoire selection. This was associated with increased interindividual microbiota variation and altered proportions of two taxa in the ileum where MHC class II on IECs is highest. Intestinal mononuclear phagocytes (MNPs) have similar MHC class II transcription but less surface MHC class II and are capable of acquiring MHC class II from IECs. Thus, epithelial-myeloid interactions mediate development of adaptive responses to microbial antigens within the gastrointestinal tract.

Placental HTRA1 cleaves α1-antitrypsin to generate a NET-inhibitory peptide.

Neutrophil extracellular traps (NETs) are important components of innate immunity. Neonatal neutrophils (polymorphonuclear leukocytes [PMNs]) fail to form NETs due to circulating NET-inhibitory peptides (NIPs), cleavage fragments of α1-antitrypsin (A1AT). How fetal and neonatal blood NIPs are generated remains unknown, however. The placenta expresses high-temperature requirement serine protease A1 (HTRA1) during fetal development, which can cleave A1AT. We hypothesized that placentally expressed HTRA1 regulates the formation of NIPs and that NET competency changed in PMNs isolated from neonatal HTRA1 knockout mice (HTRA1-/-). We found that umbilical cord blood plasma has elevated HTRA1 levels compared with adult plasma and that recombinant and placenta-eluted HTRA1 cleaves A1AT to generate an A1AT cleavage fragment (A1ATM383S-CF) of molecular weight similar to previously identified NIPs that block NET formation by adult neutrophils. We showed that neonatal mouse pup plasma contains A1AT fragments that inhibit NET formation by PMNs isolated from adult mice, indicating that NIP generation during gestation is conserved across species. Lipopolysaccharide-stimulated PMNs isolated from HTRA1+/+ littermate control pups exhibit delayed NET formation after birth. However, plasma from HTRA1-/- pups had no detectable NIPs, and PMNs from HTRA1-/- pups became NET competent earlier after birth compared with HTRA1+/+ littermate controls. Finally, in the cecal slurry model of neonatal sepsis, A1ATM383S-CF improved survival in C57BL/6 pups by preventing pathogenic NET formation. Our data indicate that placentally expressed HTRA1 is a serine protease that cleaves A1AT in utero to generate NIPs that regulate NET formation by human and mouse PMNs.

Risk awareness during operation of analytical flow cytometers and implications throughout the COVID-19 pandemic.

The COVID-19 pandemic has brought biosafety to the forefront of many life sciences. The outbreak has compelled research institutions to re-evaluate biosafety practices and potential at-risk areas within research laboratories and more specifically within Shared Resource Laboratories (SRLs). In flow cytometry facilities, biological safety assessment encompasses known hazards based on the biological sample and associated risk group, as well as potential or unknown hazards, such as aerosol generation and instrument "failure modes." Cell sorting procedures undergo clearly defined biological safety assessments and adhere to well-established biosafety guidelines that help to protect SRL staff and users against aerosol exposure. Conversely, benchtop analyzers are considered low risk due to their low sample pressure and enclosed fluidic systems, although there is little empirical evidence to support this assumption of low risk. To investigate this, we evaluated several regions on analyzers using the Cyclex-d microsphere assay, a recently established method for cell sorter aerosol containment testing. We found that aerosol and/or droplet hazards were detected on all benchtop analyzers predominantly during operation in "failure modes." These results indicate that benchtop analytical cytometers present a more complicated set of risks than are commonly appreciated.

Estrogen-related receptor alpha directly binds to p53 and cooperatively controls colon cancer growth through the regulation of mitochondrial biogenesis and function.

BACKGROUND: Of the genes that control mitochondrial biogenesis and function, ERRα emerges as a druggable metabolic target to be exploited for cancer therapy. Of the genes mutated in cancer, TP53 remains the most elusive to target. A clear understanding of how mitochondrial druggable targets can be accessed to exploit the underlying mechanism(s) explaining how p53-deficient tumors promote cell survival remains elusive. METHODS: We performed protein-protein interaction studies to demonstrate that ERRα binds to p53. Moreover, we used gene silencing and pharmacological approaches in tandem with quantitative proteomics analysis by SWATH-MS to investigate the role of the ERRα/p53 complex in mitochondrial biogenesis and function in colon cancer. Finally, we designed in vitro and in vivo studies to investigate the possibility of targeting colon cancers that exhibit defects in p53. RESULTS: Here, we are the first to identify a direct protein-protein interaction between the ligand-binding domain (LBD) of ERRα and the C-terminal domain (CTD) of p53. ERRα binds to p53 regardless of p53 mutational status. Furthermore, we show that the ERRα and p53 complex cooperatively control mitochondrial biogenesis and function. Targeting ERRα creates mitochondrial metabolic stresses, such as production of reactive oxygen species (ROS) and mitochondrial membrane permeabilization (MMP), leading to a greater cytotoxic effect that is dependent on the presence of p53. Pharmacological inhibition of ERRα impairs the growth of p53-deficient cells and of p53 mutant patient-derived colon xenografts (PDX). CONCLUSIONS: Therefore, our data suggest that by using the status of the p53 protein as a selection criterion, the ERRα/p53 transcriptional axis can be exploited as a metabolic vulnerability.

Induction and Prevention of Gastric Cancer with Combined Helicobacter Pylori and Capsaicin Administration and DFMO Treatment, Respectively.

Gastric cancer risk evolves over time due to environmental, dietary, and lifestyle changes, including Helicobacter pylori (H. pylori) infection and consumption of hot peppers (i.e., capsaicin). H. pylori infection promotes gastric mucosal injury in the early phase of capsaicin exposure. This relationship suggests a need to investigate the mechanism of how both H. pylori infection and capsaicin contribute to gastric inflammation and lead to gastric cancer. C57-Balb/c mice were infected with the H. pylori (SS1) strain and then fed capsaicin (0.05% or 0.2 g/kg/day) or not. Consequently, tumor size and phenotype were analyzed to determine the molecular mechanism driving the shift from gastritis to stomach cancer. Moreover, we used 2-difluoromethylornithine (DFMO) in mice to prevent gastric tumorigenesis by reducing inflammation and promoting recovery of disease-free stasis. This study provides evidence showing that a combination of H. pylori infection and capsaicin consumption leads to gastric carcinogenesis mediated through interleukin-6 (IL-6) stimulation with an incidence rate of 50%. The anti-inflammatory role of DFMO highlights the injurious effect of inflammation in gastric cancer development and the need to reduce gastric inflammation for cancer prevention by inhibiting IL-6. Accordingly, preventive measures such as reduced capsaicin consumption, H. pylori clearance, and DFMO treatment may lessen gastric cancer incidence.