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1. Global Proteomic Analyses of STING‐Positive and ‐Negative Macrophages Reveal STING and Non‐STING Differentially Regulated Cellular and Molecular Pathways

   https://doi.org/10.1002/prca.201900109  

   Aryal, Uma K. ; Hedrick, Victoria ; Onyedibe, Kenneth Ikenna ; Sobreira, Tiago Jose Paschoal ; Sooreshjani, Moloud Aflaki ; Wang, Modi ; Gürsoy, Ulvi Kahraman ; Sintim, Herman O. ( March 2020 , PROTEOMICS – Clinical Applications)

   Cyclic guanosine monophosphate‐adenosine monophosphate and other bacterial‐derived cyclic di‐guanosine monophosphate or cyclic di‐adenosine monophosphate trigger innate immune responses through binding to stimulator of interferon genes (STING). Thus in chronic infection, such as in periodontitis, immune cells can be exposed to bacterial DNA and/or cyclic dinucleotides, potentially activating STING to cause inflammation. Thus far the cyclic GMP‐AMP synthase‐STING‐ TANK‐binding kinase 1 pathway has been well characterized but a global perspective of how the presence or lack of STING affect the proteome is lacking. The aim of this study is to identify macrophage proteins that are affected by STING.

   Proteins are extracted from a macrophage cell line harboring STING (RAW‐Blue ISG) as well as a STING knockout (STING KO) cell line (RAW‐Lucia ISG‐KO‐STING) and global proteomics analyses are performed.

   Proteins related to kinase and phosphatase signaling, spliceosome, terpenoid backbone biosynthesis, glycosylation, ubiquitination, and phagocytosis are affected by STING knock out.

   STING pathway in macrophages is related to the regulation of several proteins that are known as potent biomarkers of various cancers and autoimmune diseases. Moreover, the relation between STING and phagocytosis is demonstrated for the first time. Further validation studies will help identify molecules and pathways that may function as diagnostic or therapeutic targets.

    

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2. Bacterial Cyclic Dinucleotides and the cGAS–cGAMP–STING Pathway: A Role in Periodontitis?

   https://doi.org/10.3390/pathogens10060675  

   Elmanfi, Samira ; Yilmaz, Mustafa ; Ong, Wilson W. ; Yeboah, Kofi S. ; Sintim, Herman O. ; Gürsoy, Mervi ; Könönen, Eija ; Gürsoy, Ulvi K. ( June 2021 , Pathogens)

   null (Ed.)

   Host cells can recognize cytosolic double-stranded DNAs and endogenous second messengers as cyclic dinucleotides—including c-di-GMP, c-di-AMP, and cGAMP—of invading microbes via the critical and essential innate immune signaling adaptor molecule known as STING. This recognition activates the innate immune system and leads to the production of Type I interferons and proinflammatory cytokines. In this review, we (1) focus on the possible role of bacterial cyclic dinucleotides and the STING/TBK1/IRF3 pathway in the pathogenesis of periodontal disease and the regulation of periodontal immune response, and (2) review and discuss activators and inhibitors of the STING pathway as immune response regulators and their potential utility in the treatment of periodontitis. PubMed/Medline, Scopus, and Web of Science were searched with the terms “STING”, “TBK 1”, “IRF3”, and “cGAS”—alone, or together with “periodontitis”. Current studies produced evidence for using STING-pathway-targeting molecules as part of anticancer therapy, and as vaccine adjuvants against microbial infections; however, the role of the STING/TBK1/IRF3 pathway in periodontal disease pathogenesis is still undiscovered. Understanding the stimulation of the innate immune response by cyclic dinucleotides opens a new approach to host modulation therapies in periodontology. 

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3. An Infection-Tolerant Mammalian Reservoir for Several Zoonotic Agents Broadly Counters the Inflammatory Effects of Endotoxin

   https://doi.org/10.1128/mBio.00588-21  

   Balderrama-Gutierrez, Gabriela ; Milovic, Ana ; Cook, Vanessa J. ; Islam, M. Nurul ; Zhang, Youwen ; Kiaris, Hippokratis ; Belisle, John T. ; Mortazavi, Ali ; Barbour, Alan G. ( April 2021 , mBio)

   Miller, Samuel I. (Ed.)

   ABSTRACT Animals that are competent reservoirs of zoonotic pathogens commonly suffer little morbidity from the infections. To investigate mechanisms of this tolerance of infection, we used single-dose lipopolysaccharide (LPS) as an experimental model of inflammation and compared the responses of two rodents: Peromyscus leucopus , the white-footed deermouse and reservoir for the agents of Lyme disease and other zoonoses, and the house mouse Mus musculus . Four hours after injection with LPS or saline, blood, spleen, and liver samples were collected and subjected to transcriptome sequencing (RNA-seq), metabolomics, and specific reverse transcriptase quantitative PCR (RT-qPCR). Differential expression analysis was at the gene, pathway, and network levels. LPS-treated deermice showed signs of sickness similar to those of exposed mice and had similar increases in corticosterone levels and expression of interleukin 6 (IL-6), tumor necrosis factor, IL-1β, and C-reactive protein. By network analysis, the M. musculus response to LPS was characterized as cytokine associated, while the P. leucopus response was dominated by neutrophil activity terms. In addition, dichotomies in the expression levels of arginase 1 and nitric oxide synthase 2 and of IL-10 and IL-12 were consistent with type M1 macrophage responses in mice and type M2 responses in deermice. Analysis of metabolites in plasma and RNA in organs revealed species differences in tryptophan metabolism. Two genes in particular signified the different phenotypes of deermice and mice: the Slpi and Ibsp genes. Key RNA-seq findings for P. leucopus were replicated in older animals, in a systemic bacterial infection, and with cultivated fibroblasts. The findings indicate that P. leucopus possesses several adaptive traits to moderate inflammation in its balancing of infection resistance and tolerance. IMPORTANCE Animals that are natural carriers of pathogens that cause human diseases commonly manifest little or no sickness as a consequence of infection. Examples include the deermouse, Peromyscus leucopus , which is a reservoir for Lyme disease and several other disease agents in North America, and some types of bats, which are carriers of viruses with pathogenicity for humans. Mechanisms of this phenomenon of infection tolerance and entailed trade-off costs are poorly understood. Using a single injection of lipopolysaccharide (LPS) endotoxin as a proxy for infection, we found that deermice differed from the mouse ( Mus musculus ) in responses to LPS in several diverse pathways, including innate immunity, oxidative stress, and metabolism. Features distinguishing the deermice cumulatively would moderate downstream ill effects of LPS. Insights gained from the P. leucopus model in the laboratory have implications for studying infection tolerance in other important reservoir species, including bats and other types of wildlife. 

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4. Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme

   https://doi.org/10.1073/pnas.2100657118  

   Patterson, Dayna C. ; Ruiz, Myrrh Perez ; Yoon, Hyerin ; Walker, Johnnie A. ; Armache, Jean-Paul ; Yennawar, Neela H. ; Weinert, Emily E. ( September 2021 , Proceedings of the National Academy of Sciences)

   Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) fromPaenibacillus dendritiformis(DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O₂state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation ofP. dendritiformisin response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities.

    

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5. Advanced age protects resistance arteries of mouse skeletal muscle from oxidative stress through attenuating apoptosis induced by hydrogen peroxide

   https://doi.org/10.1113/JP278255  

   Norton, Charles E. ; Sinkler, Shenghua Y. ; Jacobsen, Nicole L. ; Segal, Steven S. ( July 2019 , The Journal of Physiology)

   Vascular oxidative stress increases with advancing age.

   We hypothesized that resistance vessels develop resilience to oxidative stress to protect functional integrity and tested this hypothesis by exposing isolated pressurized superior epigastric arteries (SEAs) of old and young mice to H₂O₂.

   H₂O₂‐induced death was greater in smooth muscle cells (SMCs) than endothelial cells (ECs) and lower in SEAs from oldvs. young mice; the rise in vessel wall [Ca²⁺]_iinduced by H₂O₂was attenuated with ageing, as was the decline in noradrenergic vasoconstriction; genetic deletion of IL‐10 mimicked the effects of advanced age on cell survival.

   Inhibiting NO synthase or scavenging peroxynitrite reduced SMC death; endothelial denudation or inhibiting gap junctions increased SMC death; delocalization of cytochrome C activated caspases 9 and 3 to induce apoptosis.

   Vascular cells develop resilience to H₂O₂during ageing by preventing Ca²⁺overload and endothelial integrity promotes SMC survival.

   Advanced age is associated with elevated oxidative stress and can protect the endothelium from cell death induced by H₂O₂. Whether such protection occurs for intact vessels or differs between smooth muscle cell (SMC) and endothelial cell (EC) layers is unknown. We tested the hypothesis that ageing protects SMCs and ECs during acute exposure to H₂O₂(200 µm, 50 min). Mouse superior epigastric arteries (SEAs; diameter, ∼150 µm) were isolated and pressurized to 100 cmH₂O at 37˚C. For SEAs from young (4 months) mice, H₂O₂killed 57% of SMCs and 11% of ECs in malesvs. 8% and 2%, respectively, in females. Therefore, SEAs from males were studied to resolve the effect of ageing and experimental interventions. For old (24 months) mice, SMC death was reduced to 10% with diminished accumulation of [Ca²⁺]_iin the vessel wall during H₂O₂exposure. In young mice, genetic deletion of IL‐10 mimicked the protective effect of ageing on cell death and [Ca²⁺]_iaccumulation. Whereas endothelial denudation or gap junction inhibition (carbenoxolone; 100 µm) increased SMC death, inhibiting NO synthase (l‐NAME, 100 µm) or scavenging peroxynitrite (FeTPPS, 5 µm) reduced SMC death along with [Ca²⁺]_i. Despite NO toxicity via peroxynitrite formation, endothelial integrity protects SMCs. Caspase inhibition (Z‐VAD‐FMK, 50 µm) attenuated cell death with immunostaining for annexin V, cytochrome C, and caspases 3 and 9 pointing to induction of intrinsic apoptosis during H₂O₂exposure. We conclude that advanced age reduces Ca²⁺influx that triggers apoptosis, thereby promoting resilience of the vascular wall during oxidative stress.

    

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