Creatine maintains intestinal homeostasis and protects against colitis

Creatine, a nitrogenous organic acid, replenishes cytoplasmic ATP at the expense of mitochondrial ATP via the phosphocreatine shuttle. Creatine levels are maintained by diet and endogenous synthesis from arginine and glycine. Glycine amidinotransferase (GATM) catalyzes the rate-limiting step of creatine biosynthesis: the transfer of an amidino group from arginine to glycine to form ornithine and guanidinoacetate. We screened 36,530 third-generation germline mutant mice derived from N-ethyl-N-nitrosourea–mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). Among 27 colitis susceptibility phenotypes identified and mapped, one was strongly correlated with a missense mutation in Gatm in a recessive model of inheritance, and causation was confirmed by CRISPR/Cas9 gene targeting. Supplementation of homozygous Gatm mutants with exogenous creatine ameliorated the colitis phenotype. CRISPR/Cas9-targeted (Gatmc/c) mice displayed a normal peripheral immune response and immune cell homeostasis. However, the intestinal epithelium of the Gatmc/c mice displayed increased cell death and decreased proliferation during DSS treatment. In addition, Gatmc/c colonocytes showed increased metabolic stress in response to DSS with higher levels of phospho-AMPK and lower levels of phosphorylation of mammalian target of rapamycin (phospho-mTOR). These findings establish an in vivo requirement for rapid replenishment of cytoplasmic ATP within colonic epithelial cells in the maintenance of the mucosal barrier after injury.

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Cdx2 regulates immune cell infiltration in the intestine

Scientific Reports ◽

10.1038/s41598-021-95412-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Simon Chewchuk ◽

Sanzida Jahan ◽

David Lohnes

Keyword(s):

Intestinal Epithelium ◽

Target Genes ◽

Immune Cell ◽

Cell Number ◽

Macrophage Infiltration ◽

Intestinal Homeostasis ◽

Chronic Inflammatory Response ◽

Complex Protein ◽

Inflammatory Phenotype

AbstractThe intestinal epithelium is a unique tissue, serving both as a barrier against pathogens and to conduct the end digestion and adsorption of nutrients. As regards the former, the intestinal epithelium contains a diverse repertoire of immune cells, including a variety of resident lymphocytes, macrophages and dendritic cells. These cells serve a number of roles including mitigation of infection and to stimulate regeneration in response to damage. The transcription factor Cdx2, and to a lesser extent Cdx1, plays essential roles in intestinal homeostasis, and acts as a context-dependent tumour suppressor in colorectal cancer. Deletion of Cdx2 from the murine intestinal epithelium leads to macrophage infiltration resulting in a chronic inflammatory response. However the mechanisms by which Cdx2 loss evokes this response are poorly understood. To better understand this relationship, we used a conditional mouse model lacking all intestinal Cdx function to identify potential target genes which may contribute to this inflammatory phenotype. One such candidate encodes the histocompatability complex protein H2-T3, which functions to regulate intestinal iCD8α lymphocyte activity. We found that Cdx2 occupies the H3-T3 promoter in vivo and directly regulates its expression via a Cdx response element. Loss of Cdx function leads to a rapid and pronounced attenuation of H2-T3, followed by a decrease in iCD8α cell number, an increase in macrophage infiltration and activation of pro-inflammatory cascades. These findings suggest a previously unrecognized role for Cdx in intestinal homeostasis through H2-T3-dependent regulation of iCD8α cells.

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The amino acid sensor GCN2 inhibits inflammatory responses to apoptotic cells promoting tolerance and suppressing systemic autoimmunity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1504276112 ◽

2015 ◽

Vol 112 (34) ◽

pp. 10774-10779 ◽

Cited By ~ 58

Author(s):

Buvana Ravishankar ◽

Haiyun Liu ◽

Rahul Shinde ◽

Kapil Chaudhary ◽

Wei Xiao ◽

...

Keyword(s):

Cell Activation ◽

Immune Cell ◽

Inflammatory Responses ◽

Apoptotic Cell ◽

Metabolic Stress ◽

Tolerance Induction ◽

Apoptotic Cells ◽

Cell Clearance

Efficient apoptotic cell clearance and induction of immunologic tolerance is a critical mechanism preventing autoimmunity and associated pathology. Our laboratory has reported that apoptotic cells induce tolerance by a mechanism dependent on the tryptophan catabolizing enzyme indoleamine 2,3 dioxygenase 1 (IDO1) in splenic macrophages (MΦ). The metabolic-stress sensing protein kinase GCN2 is a primary downstream effector of IDO1; thus, we tested its role in apoptotic cell-driven immune suppression. In vitro, expression of IDO1 in MΦs significantly enhanced apoptotic cell-driven IL-10 and suppressed IL-12 production in a GCN2-dependent mechanism. Suppression of IL-12 protein production was due to attenuation of IL-12 mRNA association with polyribosomes inhibiting translation while IL-10 mRNA association with polyribosomes was not affected. In vivo, apoptotic cell challenge drove a rapid, GCN2-dependent stress response in splenic MΦs with increased IL-10 and TGF-β production, whereas myeloid-specific deletion of GCN2 abrogated regulatory cytokine production with provocation of inflammatory T-cell responses to apoptotic cell antigens and failure of long-tolerance induction. Consistent with a role in prevention of apoptotic cell driven autoreactivity, myeloid deletion of GCN2 in lupus-prone mice resulted in increased immune cell activation, humoral autoimmunity, renal pathology, and mortality. In contrast, activation of GCN2 with an agonist significantly reduced anti-DNA autoantibodies and protected mice from disease. Thus, this study implicates a key role for GCN2 signals in regulating the tolerogenic response to apoptotic cells and limiting autoimmunity.

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Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis

Nature Communications ◽

10.1038/s41467-021-25457-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Arthur C. Sletten ◽

Jessica W. Davidson ◽

Busra Yagabasan ◽

Samantha Moores ◽

Michaela Schwaiger-Haber ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Cultured Cells ◽

Mammalian Target Of Rapamycin ◽

Metabolic Stress ◽

Cell Dysfunction ◽

Regulation Of Metabolism ◽

Signaling Axis ◽

Nucleolar Rna

AbstractDyslipidemia and resulting lipotoxicity are pathologic signatures of metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not well understood. Herein, we show that disruption of the box H/ACA SNORA73 small nucleolar RNAs encoded within the small nucleolar RNA hosting gene 3 (Snhg3) causes resistance to lipid-induced cell death and general oxidative stress in cultured cells. This protection from metabolic stress is associated with broad reprogramming of oxidative metabolism that is dependent on the mammalian target of rapamycin signaling axis. Furthermore, we show that knockdown of SNORA73 in vivo protects against hepatic steatosis and lipid-induced oxidative stress and inflammation. Our findings demonstrate a role for SNORA73 in the regulation of metabolism and lipotoxicity.

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NSAIDs disrupt intestinal homeostasis by suppressing macroautophagy in intestinal epithelial cells

Scientific Reports ◽

10.1038/s41598-019-51067-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Ana M. Chamoun-Emanuelli ◽

Laura K. Bryan ◽

Noah D. Cohen ◽

Taylor L. Tetrault ◽

Joseph A. Szule ◽

...

Keyword(s):

Inflammatory Response ◽

Clinical Manifestations ◽

Building Blocks ◽

Mucosal Barrier ◽

High Morbidity ◽

Intestinal Damage ◽

Intestinal Epithelial ◽

Intestinal Homeostasis

Abstract Small intestinal damage induced by nonsteroidal anti-inflammatory drugs (NSAIDs) remains an under-recognized clinical disorder. The incomplete understanding of the pathophysiology has hampered the development of prevention and treatment strategies leading to the high morbidity and mortality rates. NSAIDs are known to modulate macroautophagy, a process indispensable for intestinal homeostasis. Whether NSAIDs stimulate or repress macroautophagy and how this correlates with the clinical manifestations of NSAID enteropathy, however, remains unknown. The objectives of this study were to determine whether NSAIDs impaired macroautophagy and how this affects macroautophagy-regulated intestinal epithelial cell (IEC) processes essential for intestinal homeostasis (i.e., clearance of invading pathogens, secretion and composition of mucus building blocks, and inflammatory response). We show that NSAID treatment of IECs inhibits macroautophagy in vitro and in vivo. This inhibition was likely attributed to a reduction in the area and/or distribution of lysosomes available for degradation of macroautophagy-targeted cargo. Importantly, IEC regulatory processes necessary for intestinal homeostasis and dependent on macroautophagy were dysfunctional in the presence of NSAIDs. Since macroautophagy is essential for gastrointestinal health, NSAID-induced inhibition of macroautophagy might contribute to the severity of intestinal injury by compromising the integrity of the mucosal barrier, preventing the clearance of invading microbes, and exacerbating the inflammatory response.

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Dynamics of hepatic and peripheral insulin effects suggest common rate-limiting step in vivo

Diabetes ◽

10.2337/diabetes.42.2.296 ◽

1993 ◽

Vol 42 (2) ◽

pp. 296-306 ◽

Cited By ~ 9

Author(s):

D. C. Bradley ◽

R. A. Poulin ◽

R. N. Bergman

Keyword(s):

Rate Limiting Step ◽

Limiting Step ◽

Rate Limiting

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Adenosine A2A receptor signaling promotes FoxO associated autophagy in chondrocytes

Scientific Reports ◽

10.1038/s41598-020-80244-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Benjamin Friedman ◽

Carmen Corciulo ◽

Cristina M. Castro ◽

Bruce N. Cronstein

Keyword(s):

Cell Line ◽

Metabolic Stress ◽

Human Cell Line ◽

Adenosine A2a Receptor ◽

Autophagic Flux ◽

A2a Receptor ◽

Adenosine A2a ◽

A2ar Agonist

AbstractAutophagy, a homeostatic pathway upregulated during cellular stress, is decreased in osteoarthritic chondrocytes and this reduction in autophagy is thought to contribute to the development and progression of osteoarthritis (OA). The adenosine A2A receptor (A2AR) is a potent anti-inflammatory receptor and deficiency of this receptor leads to the development of OA in mice. Moreover, treatment using liposomally conjugated adenosine or a specific A2AR agonist improved joint scores significantly in both rats with post-traumatic OA (PTOA) and mice subjected to a high fat diet obesity induced OA. Importantly, A2AR ligation is beneficial for mitochondrial health and metabolism in vitro in primary and the TC28a2 human cell line. An additional set of metabolic, stress-responsive, and homeostatic mediators include the Forkhead box O transcription factors (FoxOs). Data has shown that mouse FoxO knockouts develop early OA with reduced cartilage autophagy, indicating that FoxO-induced homeostasis is important for articular cartilage. Given the apparent similarities between A2AR and FoxO signaling, we tested the hypothesis that A2AR stimulation improves cartilage function through activation of the FoxO proteins leading to increased autophagy in chondrocytes. We analyzed the signaling pathway in the human TC28a2 cell line and corroborated these findings in vivo in a metabolically relevant obesity-induced OA mouse model. We found that A2AR stimulation increases activation and nuclear localization of FoxO1 and FoxO3, promotes an increase in autophagic flux, improves metabolic function in chondrocytes, and reduces markers of apoptosis in vitro and reduced apoptosis by TUNEL assay in vivo. A2AR ligation additionally enhances in vivo activation of FoxO1 and FoxO3 with evidence of enhanced autophagic flux upon injection of the liposome-associated A2AR agonist in a mouse obesity-induced OA model. These findings offer further evidence that A2AR may be an excellent target for promoting chondrocyte and cartilage homeostasis.

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Aη-α and Aη-β peptides impair LTP ex vivo within the low nanomolar range and impact neuronal activity in vivo

Alzheimer s Research & Therapy ◽

10.1186/s13195-021-00860-1 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Maria Mensch ◽

Jade Dunot ◽

Sandy M. Yishan ◽

Samuel S. Harris ◽

Aline Blistein ◽

...

Keyword(s):

Neuronal Activity ◽

Ex Vivo ◽

Long Term Potentiation ◽

Network Activity ◽

Strongly Correlated ◽

App Processing ◽

Term Potentiation ◽

Hippocampal Network

Abstract Background Amyloid precursor protein (APP) processing is central to Alzheimer’s disease (AD) etiology. As early cognitive alterations in AD are strongly correlated to abnormal information processing due to increasing synaptic impairment, it is crucial to characterize how peptides generated through APP cleavage modulate synapse function. We previously described a novel APP processing pathway producing η-secretase-derived peptides (Aη) and revealed that Aη–α, the longest form of Aη produced by η-secretase and α-secretase cleavage, impaired hippocampal long-term potentiation (LTP) ex vivo and neuronal activity in vivo. Methods With the intention of going beyond this initial observation, we performed a comprehensive analysis to further characterize the effects of both Aη-α and the shorter Aη-β peptide on hippocampus function using ex vivo field electrophysiology, in vivo multiphoton calcium imaging, and in vivo electrophysiology. Results We demonstrate that both synthetic peptides acutely impair LTP at low nanomolar concentrations ex vivo and reveal the N-terminus to be a primary site of activity. We further show that Aη-β, like Aη–α, inhibits neuronal activity in vivo and provide confirmation of LTP impairment by Aη–α in vivo. Conclusions These results provide novel insights into the functional role of the recently discovered η-secretase-derived products and suggest that Aη peptides represent important, pathophysiologically relevant, modulators of hippocampal network activity, with profound implications for APP-targeting therapeutic strategies in AD.

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Non-radioactive imaging strategies for in vivo immune cell tracking

Physical Sciences Reviews ◽

10.1515/psr-2020-0205 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Łukasz Kiraga ◽

Paulina Kucharzewska ◽

Damian Strzemecki ◽

Tomasz P. Rygiel ◽

Magdalena Król

Keyword(s):

Diagnostic Imaging ◽

Contrast Agents ◽

Cell Tracking ◽

Immune Cell ◽

Imaging Techniques ◽

Diagnostic Technique ◽

Cell Labeling ◽

X Ray Imaging ◽

Disease Study

Abstract In vivo tracking of administered cells chosen for specific disease treatment may be conducted by diagnostic imaging techniques preceded by cell labeling with special contrast agents. The most commonly used agents are those with radioactive properties, however their use in research is often impossible. This review paper focuses on the essential aspect of cell tracking with the exclusion of radioisotope tracers, therefore we compare application of different types of non-radioactive contrast agents (cell tracers), methods of cell labeling and application of various techniques for cell tracking, which are commonly used in preclinical or clinical studies. We discuss diagnostic imaging methods belonging to three groups: (1) Contrast-enhanced X-ray imaging, (2) Magnetic resonance imaging, and (3) Optical imaging. In addition, we present some interesting data from our own research on tracking immune cell with the use of discussed methods. Finally, we introduce an algorithm which may be useful for researchers planning leukocyte targeting studies, which may help to choose the appropriate cell type, contrast agent and diagnostic technique for particular disease study.

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The Role of Melatonin on NLRP3 Inflammasome Activation in Diseases

Antioxidants ◽

10.3390/antiox10071020 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1020

Author(s):

Burak Ibrahim Arioz ◽

Emre Tarakcioglu ◽

Melis Olcum ◽

Sermin Genc

Keyword(s):

Nlrp3 Inflammasome ◽

Intracellular Signaling ◽

Metabolic Diseases ◽

Metabolic Stress ◽

Clinical Importance ◽

Rapid Identification ◽

In Vivo Studies ◽

Inflammasome Activation

NLRP3 inflammasome is a part of the innate immune system and responsible for the rapid identification and eradication of pathogenic microbes, metabolic stress products, reactive oxygen species, and other exogenous agents. NLRP3 inflammasome is overactivated in several neurodegenerative, cardiac, pulmonary, and metabolic diseases. Therefore, suppression of inflammasome activation is of utmost clinical importance. Melatonin is a ubiquitous hormone mainly produced in the pineal gland with circadian rhythm regulatory, antioxidant, and immunomodulatory functions. Melatonin is a natural product and safer than most chemicals to use for medicinal purposes. Many in vitro and in vivo studies have proved that melatonin alleviates NLRP3 inflammasome activity via various intracellular signaling pathways. In this review, the effect of melatonin on the NLRP3 inflammasome in the context of diseases will be discussed.

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A Set of Cell Lines Derived from a Genetic Murine Glioblastoma Model Recapitulates Molecular and Morphological Characteristics of Human Tumors

Cancers ◽

10.3390/cancers13020230 ◽

2021 ◽

Vol 13 (2) ◽

pp. 230

Author(s):

Barbara Costa ◽

Michael N.C. Fletcher ◽

Pavle Boskovic ◽

Ekaterina L. Ivanova ◽

Tanja Eisemann ◽

...

Keyword(s):

Cell Lines ◽

Immune Cell ◽

Treatment Options ◽

Morphological Characteristics ◽

Molecular Heterogeneity ◽

Double Knockout ◽

In Vivo Models ◽

Human Glioblastoma ◽

Human Gbm

Glioblastomas (GBM) are the most aggressive tumors affecting the central nervous system in adults, causing death within, on average, 15 months after diagnosis. Immunocompetent in-vivo models that closely mirror human GBM are urgently needed for deciphering glioma biology and for the development of effective treatment options. The murine GBM cell lines currently available for engraftment in immunocompetent mice are not only exiguous but also inadequate in representing prominent characteristics of human GBM such as infiltrative behavior, necrotic areas, and pronounced tumor heterogeneity. Therefore, we generated a set of glioblastoma cell lines by repeated in vivo passaging of cells isolated from a neural stem cell-specific Pten/p53 double-knockout genetic mouse brain tumor model. Transcriptome and genome analyses of the cell lines revealed molecular heterogeneity comparable to that observed in human glioblastoma. Upon orthotopic transplantation into syngeneic hosts, they formed high-grade gliomas that faithfully recapitulated the histopathological features, invasiveness and immune cell infiltration characteristic of human glioblastoma. These features make our cell lines unique and useful tools to study multiple aspects of glioblastoma pathomechanism and to test novel treatments in an intact immune microenvironment.

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