scholarly journals Skap2 Regulates Atherosclerosis through Macrophage Polarization and Efferocytosis

2019 ◽  
Author(s):  
Danielle Hyatt ◽  
Allison E. Schroeder ◽  
Ashita Bhatnagar ◽  
David E. Golan ◽  
Kenneth D. Swanson ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Macrophage Polarization ◽  
Polarization State ◽  
Critical Role ◽  
Adaptor Protein ◽  
Foam Cells ◽  
Standard Diet ◽  
Macrophage Phagocytosis ◽  
Human Atheroma

ABSTRACTRationaleAtherosclerosis causes more deaths than any other pathophysiologic process. It has a well-established inflammatory, macrophage-mediated component, but important and potentially protective intracellular macrophage processes in atherosclerosis remain enigmatic. Src Kinase-Associated Phosphoprotein 2 (Skap2) is a macrophage-predominant adaptor protein critical for cytoskeletal reorganization, and thereby, for macrophage migration and chemotaxis. The role of macrophage Skap2 in atherosclerosis is unknown and deserves exploration.ObjectiveTo establish the critical role of Skap2 in macrophage-mediated atherosclerotic plaque homeostasis.ResultsIn human arterial gene expression analysis, Skap2 expression is enriched in macrophage-containing areas of human atheroma, and the transcript level varies with plaque characteristics. We have discovered that deletion of Skap2 accelerates atherosclerosis by threefold in ApoE-/- mice on standard diet. Skap2 expression is switched on only as monocytes differentiate into macrophages, so Skap2-/- monocytes have no defect in infiltrating the atheroma. On the other hand, once they fully differentiate, Skap2-deficient macrophages cannot polarize efficiently into alternatively-activated, regulatory cells, and instead they preferentially polarize toward the classical pro-inflammatory phenotype both ex vivo and within the developing atheroma. This defect extends to polarized effector functions, as ex vivo analysis of macrophage phagocytosis of dying foam cells indicates that Skap2 is required for the regulatory process of efferocytosis.ConclusionsTaken together, our findings support a model in which Skap2 drives a regulatory, efferocytic mode of behavior to quell atherosclerosis.CONDENSED ABSTRACT / SUMMARYSkap2—a macrophage protein found in the human atheroma—is atheroprotective. Skap2-null mice, whose foam cells do not migrate well due to a defect in integrin-induced cytoskeletal rearrangement, have accelerated atherosclerosis. Skap2 is not expressed in monocytes but becomes important once they reach the atheroma and become macrophage foam cells, at which point it drives toward a regulatory, anti-inflammatory polarization state required for efficient efferocytosis of dying foam cells. Thus, Skap2 drives a protective, regulatory mode of behavior, supporting the fact that macrophages are not solely deleterious in atherosclerosis, and further pointing to efferocytosis as a target for therapy.There are no relationships to disclose.

scholarly journals Enhancement of Methacholine-Evoked Tracheal Contraction Induced by Bacterial Lipopolysaccharides Depends on Epithelium and Tumor Necrosis Factor

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
T. Secher ◽  
F. Rodrigues Coelho ◽  
N. Noulin ◽  
A. Lino dos Santos Franco ◽  
V. Quesniaux ◽  
...  
Keyword(s):  
Contractile Response ◽  
Ex Vivo ◽  
Adaptor Protein ◽  
Respiratory Pattern ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Bacterial Lipopolysaccharides ◽  
Necrosis Factor

Inhaled bacterial lipopolysaccharides (LPSs) induce an acute tumour necrosis factor-alpha (TNF-α-) dependent inflammatory response in the murine airways mediated by Toll-like receptor 4 (TLR4) via the myeloid differentiation MyD88 adaptor protein pathway. However, the contractile response of the bronchial smooth muscle and the role of endogenous TNFα in this process have been elusive. We determined the in vivo respiratory pattern of C57BL/6 mice after intranasal LPS administration with or without the presence of increasing doses of methacholine (MCh). We found that LPS administration altered the basal and MCh-evoked respiratory pattern that peaked at 90 min and decreased thereafter in the next 48 h, reaching basal levels 7 days later. We investigated in controlled ex vivo condition the isometric contraction of isolated tracheal rings in response to MCh cholinergic stimulation. We observed that preincubation of the tracheal rings with LPS for 90 min enhanced the subsequent MCh-induced contractile response (hyperreactivity), which was prevented by prior neutralization of TNFα with a specific antibody. Furthermore, hyperreactivity induced by LPS depended on an intact epithelium, whereas hyperreactivity induced by TNFα was well maintained in the absence of epithelium. Finally, the enhanced contractile response to MCh induced by LPS when compared with control mice was not observed in tracheal rings from TLR4- or TNF- or TNF-receptor-deficient mice. We conclude that bacterial endotoxin-mediated hyperreactivity of isolated tracheal rings to MCh depends upon TLR4 integrity that signals the activation of epithelium, which release endogenous TNFα.

scholarly journals Defective liver glycogen autophagy related to hyperinsulinemia in intrauterine growth-restricted newborn wistar rats

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Juan de Toro-Martín ◽  
Tamara Fernández-Marcelo ◽  
Águeda González-Rodríguez ◽  
Fernando Escrivá ◽  
Ángela M. Valverde ◽  
...  
Keyword(s):  
Wistar Rats ◽  
Metabolic Diseases ◽  
Critical Role ◽  
Liver Glycogen ◽  
Hormonal Control ◽  
Standard Diet ◽  
Major Organ ◽  
Mtorc1 Pathway

Abstract Maternal malnutrition plays a critical role in the developmental programming of later metabolic diseases susceptibility in the offspring, such as obesity and type 2 diabetes. Because the liver is the major organ that produces and supplies blood glucose, we aimed at defining the potential role of liver glycogen autophagy in the programming of glucose metabolism disturbances. To this end, newborns were obtained from pregnant Wistar rats fed ad libitum with a standard diet or 65% food-restricted during the last week of gestation. We found that newborns from undernourished mothers showed markedly high basal insulin levels whereas those of glucagon were decreased. This unbalance led to activation of the mTORC1 pathway and inhibition of hepatic autophagy compromising the adequate handling of glycogen in the very early hours of extrauterine life. Restoration of autophagy with rapamycin but not with glucagon, indicated no defect in autophagy machinery per se, but in signals triggered by glucagon. Taken together, these results support the notion that hyperinsulinemia is an important mechanism by which mobilization of liver glycogen by autophagy is defective in food-restricted animals. This early alteration in the hormonal control of liver glycogen autophagy may influence the risk of developing metabolic diseases later in life.

Development of an ex vivo cellular model of rheumatoid arthritis: Critical role of cd14-positive monocyte/macrophages in the development of pannus tissue

2007 ◽  
Vol 56 (9) ◽  
pp. 2875-2885 ◽  
Author(s):  
Toshiko Nozaki ◽  
Kyoko Takahashi ◽  
Osamu Ishii ◽  
Sachio Endo ◽  
Kyoji Hioki ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Ex Vivo ◽  
Critical Role ◽  
Cellular Model ◽  
Pannus Tissue

Effects of diesel exhaust particles on macrophage polarization

2016 ◽  
Vol 36 (4) ◽  
pp. 412-420 ◽  
Author(s):  
N Labranche ◽  
C El Khattabi ◽  
G Berkenboom ◽  
S Pochet
Keyword(s):  
Mrna Expression ◽  
Diesel Exhaust ◽  
Macrophage Polarization ◽  
Critical Role ◽  
Diesel Exhaust Particles ◽  
Western Blots ◽  
Cardiovascular Morbidity And Mortality ◽  
Tnf Α ◽  
Exhaust Particles

Background: Exposure to diesel exhaust particles (DEP) has long been associated with increased cardiovascular morbidity and mortality. The development of DEP toxicity seems to be linked to inflammation in which macrophages play a critical role. Macrophages can be polarized into proinflammatory M1 or anti-inflammatory M2 macrophages. The aim of this study was to identify the role of inflammation in DEP-induced toxicity by assessing the effects of DEP on macrophage polarization. Methods: Monocyte-derived macrophages (Mϕ) were stimulated with interferon γ and lipopolysaccharide or interleukin (IL)-4 to obtain M1 and M2 subtypes, respectively. To test the polarization capacity of DEP, Mϕ cells were exposed to DEP and compared to Mϕ, M1, and M2. We also studied the effects of DEP on already-polarized M1 or M2. The M1 markers assessed were tumor necrosis factor α (TNF-α) and IL-1β, while the M2 markers were the mannose receptor C type 1 (MRC-1) and transglutaminase 2 (TGM2). Results: Western blots revealed a 31 kDa band corresponding to pro-IL-1β, but only in M1-polarized macrophages. In M1, we also observed an upregulation of TNF-α messenger RNA (mRNA) expression. MRC-1 and TGM2 mRNA expression were only significantly enhanced in M2. DEP had no effect on any of the M1/M2 markers assessed. Moreover, DEP were not able to modify the phenotype of already-polarized M1 or M2. Conclusion: Mϕ incubation with DEP did not have any effect on macrophage polarization, at least on the markers assessed in this study, namely, TNF-α/IL-1β for M1, and MRC-1/TGM2 for M2. Hence, these data argue against an important role of inflammation in DEP-induced vascular toxicity.

scholarly journals S-Palmitoylation determines TMEM55B-dependent positioning of lysosomes

2021 ◽  
Author(s):  
Sönke Rudnik ◽  
Saskia Heybrock ◽  
Paul Saftig ◽  
Markus Damme
Keyword(s):  
Molecular Motors ◽  
Protein Complexes ◽  
Critical Role ◽  
Adaptor Protein ◽  
Retrograde Transport ◽  
Microtubule Motors ◽  
Lysosomal Sorting ◽  
Sorting Motif ◽  
Spatio Temporal

The spatio-temporal cellular distribution of lysosomes depends on active transport mainly driven by microtubule-motors such as kinesins and dynein. Different protein complexes attach these molecular motors to their vesicular cargo: TMEM55B, as an integral lysosomal membrane protein, is a component of such a complex mediating the retrograde transport of lysosomes by establishing an interaction with the cytosolic scaffold protein JIP4 and dynein/dynactin. Here we show that TMEM55B and its paralog TMEM55A are S-palmitoylated proteins and lipidated at multiple cysteine-residues. Mutation of all cysteines in TMEM55B prevents S-palmitoylation and causes the retention of the mutated protein in the Golgi-apparatus. Consequently, non-palmitoylated TMEM55B is no longer able to modulate lysosomal positioning and the perinuclear clustering of lysosomes. Additional mutagenesis of the dileucine-based lysosomal sorting motif in non-palmitoylated TMEM55B leads to partial missorting to the plasma membrane instead of retention in the Golgi, implicating a direct effect of S-palmitoylation on the adaptor-protein-dependent sorting of TMEM55B. Our data suggest a critical role of S-palmitoylation on the trafficking of TMEM55B and TMEM55B-dependent lysosomal positioning.

scholarly journals Critical role of the Toll-like receptor signal adaptor protein MyD88 in acute allograft rejection

2003 ◽  
Vol 111 (10) ◽  
pp. 1571-1578 ◽  
Author(s):  
Daniel R. Goldstein ◽  
Bethany M. Tesar ◽  
Shizuo Akira ◽  
Fadi G. Lakkis
Keyword(s):  
Allograft Rejection ◽  
Critical Role ◽  
Adaptor Protein ◽  
Toll Like Receptor ◽  
Acute Allograft Rejection ◽  
Receptor Signal

The Role of Microtubule Regulatory Protein Stathmin (STMN1) in Megakaryopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4591-4591
Author(s):  
Camelia Iancu-Rubin ◽  
Joseph Tripodi ◽  
Vesna Najfeld ◽  
George F. Atweh
Keyword(s):  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Critical Role ◽  
Regulatory Protein ◽  
Active Form ◽  
Fish Analysis ◽  
Ploidy Levels ◽  
Significant Difference

Abstract Abstract 4591 Megakaryopoiesis is a complex process in which hematopoietic progenitor cells proliferate and acquire megakaryocyte (MK)-specific markers then undergo polyploidization (i.e. acquisition of DNA content >2n) and cytoplasmic maturation, and start producing platelets. Polyploidization and platelet formation are highly dependent on microtubule (MT) function. To become polyploid, MK undergo abortive mitosis that is mediated by a mitotic spindle that consists of MT. Mature polyploid MK extend cytoplasmic extensions (i.e. proplatelets) into the vascular space and release platelets into the circulation. MT provide the structural scaffold for the proplatelets and mediate the transport of organelles and specific granules into nascent platelets. Despite the critical role of MT in MK biology, the regulation of MT in MK is poorly defined. Stathmin (STMN1) is a cytosolic phosphoprotein whose major function is to regulate MT function by promoting their depolymerization. We had previously shown that STMN1 is expressed at high levels early during megakaryopoiesis and is downregulated later during MK maturation. We also showed that inhibition of STMN1 expression increased ploidy while its overexpression decreased ploidy of MK-like cell lines. Thus, we hypothesized that the dynamic regulation of STMN1 expression may be necessary for megakaryopoiesis and that perturbing its expression may impair MK polyploidization and platelet production. To test this hypothesis, we developed feline immunodeficiency virus (FIV)-based lentiviruses that express STMN1 to investigate the effects of overexpression in primary MK. Since the depolymerizing activity of STMN1 can be inactivated by a variety of cellular kinases, we generated a STMN1 vectors that expresses wild-type (WT) and another that expresses a contitutively active phosphorylation-deficient mutant of STMN1 (MT). We also developed a vector that expresses GFP as a negative control. Human MK generated ex vivo in liquid culture from CD34+ cells were infected with these different lentiviruses. After ectopic STMN1 expression by RT-PCR and flow cytometry was confirmed, MK differentiation was assessed in the presence or absence of STMN1 overexpression. Uninfected MK and MK infected with GFP lentiviruses differentiated and matured into large, easily recognizable cells with typical nuclear morphology and expressed similar levels of CD41 and CD42b by flow cytometry. The numbers of MK generated in the presence of WT-STMN1 expressing lentiviruses was similar to that generated in the cultures infected with control lentiviruses, while the number of MK generated in the presence of phosphorylation-deficient MT-STMN1 was drasticaly reduced. Similarly, the numbers of CD41+ and CD42b+ MK generated in the presence of MT-STMN1 was reduced two and three times, respectively, suggesting that overexpression of a contitutively active form of STMN1 prevents MK differentiation and maturation. We then evaluated the effects of STMN1 overexpression on MK polyploidization by determining the number X and Y chromosomes by FISH analysis. While a normal diploid cell has one copy of each chromosome, cells with ploidy levels of 4N, 8N and 16N will have 2, 4 and 8 copies, respectively. There was no significant difference between the fraction of polyploid MK infected with control-GFP and those infected with WT-STMN1 lentiviruses. In contrast, the fraction of polyploid MK infected with MT-STMN1 lentiviruses was reduced by approximately 50%, suggesting that STMN1 overexpression impairs the ability of MK to become polyploid. In conclusion, we demonstrated that perturbing the normal downregulation of STMN1 in primary human MK impairs differentiation and polyploidization. Since STMN1 is expressesd at extremely high levels in a variety of human leukemias, we have started assessing STMN1 expression expression in patients with hematological malignancies characterized by striking abnormalities in their MK lineage. Such studies might validate the role of MT regulation in MK biology in vivo and support the development of potential therapeutic strategies to target MT and/or STMN1 function in MK and platelet disorders. Disclosures: No relevant conflicts of interest to declare.

Restoration of Barrier Function in Injured Intestinal Mucosa

2007 ◽  
Vol 87 (2) ◽  
pp. 545-564 ◽  
Author(s):  
Anthony T. Blikslager ◽  
Adam J. Moeser ◽  
Jody L. Gookin ◽  
Samuel L. Jones ◽  
Jack Odle
Keyword(s):  
Small Intestine ◽  
Basement Membrane ◽  
Barrier Function ◽  
Ex Vivo ◽  
Critical Role ◽  
Acute Injury ◽  
Epithelial Basement Membrane ◽  
Mammalian Tissues ◽  
Epithelial Basement

Mucosal repair is a complex event that immediately follows acute injury induced by ischemia and noxious luminal contents such as bile. In the small intestine, villous contraction is the initial phase of repair and is initiated by myofibroblasts that reside immediately beneath the epithelial basement membrane. Subsequent events include crawling of healthy epithelium adjacent to the wound, referred to as restitution. This is a highly regulated event involving signaling via basement membrane integrins by molecules such as focal adhesion kinase and growth factors. Interestingly, however, ex vivo studies of mammalian small intestine have revealed the importance of closure of the interepithelial tight junctions and the paracellular space. The critical role of tight junction closure is underscored by the prominent contribution of the paracellular space to measures of barrier function such as transepithelial electrical resistance. Additional roles are played by subepithelial cell populations, including neutrophils, related to their role in innate immunity. The net result of reparative mechanisms is remarkably rapid closure of mucosal wounds in mammalian tissues to prevent the onset of sepsis.

scholarly journals Adrenergic/Cholinergic Immunomodulation in the Rat Model—In VivoVeritas?

1998 ◽  
Vol 6 (3-4) ◽  
pp. 245-252 ◽  
Author(s):  
I. Rinner ◽  
P. Felsner ◽  
P. M. Liebmann ◽  
D. Hofer ◽  
A. Wölfler ◽  
...  
Keyword(s):  
Rat Model ◽  
Ex Vivo ◽  
Critical Role ◽  
Thymic Epithelial Cells ◽  
Cholinergic Pathways ◽  
Culture Supernatants ◽  
T Cell Maturation ◽  
The Brain

For several years, our group has been studying thein vivorole of adrenergic and cholinergic mechanisms in the immune-neuroendocrine dialogue in the rat model. The main results of these studies can be summarized as follows: (1) exogenous or endogenous catecholamines suppress PBL functions through alpha-2-receptor-mediated mechanisms, lymphocytes of the spleen are resistant to adrenergicin vivostimulation, (2) direct or indirect cholinergic treatment leads to enhancedex vivofunctions of splenic and thymic lymphocytes leaving PBL unaffected, (3) cholinergic pathways play a critical role in the “talking back” of the immune system to the brain, (4) acetylcholine inhibits apoptosis of thymocytes possibly via direct effects on thymic epithelial cells, and may thereby influence T-cell maturation, (5) lymphocytes of the various immunological compartments were found to be equipped with the key enzymes for the synthesis of both acetylcholine and norepinephrine, and to secrete these neurotransmitters in culture supernatants

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