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Monocytic Glutaredoxin 1 Protects Mice against Obesity, Hyperglycemia and Atherosclerosis
fibronectin (FN); the latter is an important multi-functional protein. The objective of this study was to examine the differential reactivity of fenamic acid NSAIDs when oxidized by MPO and the consequent effects on FN. Studies using ESR spin trapping with DMPO revealed that N-phenanthranilic acid (N-PA) produced a stable radical when oxidized by MPO/H2O2; diphenylamine (DPA) and 3-methyldiphenylamine (3-MDA) produced ●OH (DMPO/●OH). In addition, the analogs showed the formation of GS● after the addition of GSH to the MPO/H2O2 system, with 3-MDA producing the most intense DMPO/GS● spectrum. To determine the cytotoxic potential of these radical metabolites, we used HL-60 promyelocytic leukemia cells which contain MPO. 3-MDA, DPA and N-PA demonstrated MPO-catalyzed cytotoxicity in these cells, but not mefenamic acid (MFA). LC/MS analysis of the DPA product formation showed the formation of a hydroxydiphenylamine and a quinoneimine product, indicating MPO oxidation resulted in a hydroxylation reaction. The quinoneimine products appeared to react with GSH, forming a GSH conjugate. Immuno-spin trapping analysis demonstrated that MFA was the most potent in producing FN-protein free radical formation in a MPO-system. However, FN aggregation analysis by silver staining was most prominent with DPA. MFA and its analogues showed the formation of free radicals in a peroxidase-system which interferes with normal cell functioning. Future studies are needed to determine the functional impact of fenamic acid NSAID mediated damage to FN.
doi: 10.1016/j.freeradbiomed.2016.10.136 96 Monocytic Glutaredoxin 1 Protects Mice against Obesity, Hyperglycemia and Atherosclerosis Kevin Downs1, Sina Tavakoli1, John D. Short1, Huynh Nga Nguyen1, and Reto Asmis1 1 University of Texas Health Science Center at San Antonio, USA We previously reported that glutaredoxin 1 (Grx1) protects monocytes from metabolic-stress induced priming, dysregulation and hypersensitization to chemokines. To address the role of monocytic Grx1 in mice and in the development of atherogenesis and obesity, we transplanted bone marrow (BM) from either wildtype (WT) or Grx1-/- (Grx1Leuko -/-) donor mice into atherosclerosisprone LDLR-/- mice and fed these mice a high-fat diet (HFD) for up to 20 weeks. Grx1Leuko -/- mice showed accelerated weight gain after 9 weeks followed by early onset of hyperglycemia. After 6 weeks on HFD, atherosclerotic lesions were slightly larger in Grx1Leuko -/mice than in WT, but the differences did not reach statistical significance. However, after 20 weeks, Grx1Leuko -/- mice showed 36% larger lesions than WT-BM recipients, and monocyte chemotaxis in vivo was increased 1.6-fold. Furthermore, compared to WT-BM recipients, adipose tissues and livers of Grx1Leuko -/- mice also showed increased macrophage content and elevated tissue inflammation as determined by IHC and qRT-PCR. Adipose tissue in particular, showed significant increases in the expression of proinflammatory genes in addition to an increased abundance proinflammatory “crown-like” structures. Gene expression analysis by qRT-PCR of peritoneal macrophages revealed that macrophages isolated from Grx1-/- mice also have increased expression of pro-inflammatory M1-associated genes and decreased M2-associated genes after activation with INF+TNF and IL-4 respectively. Additionally, macrophages from Grx1-/- mice exposed to metabolic stress also display increased protein-Sglutathionylation, enhanced hypersensitization to chemokine, and impaired autophagy compared to wild-type cells. Taken together, our data show that loss of monocytic Grx1 worsens monocyte priming and accelerates the infiltration of dysfunctional monocyte-
derived macrophages into tissues, such as aorta, liver and adipose tissues. We conclude that monocytic Grx1 is critical for maintaining metabolic homeostasis in mice and protects against obesity and atherosclerosis.
doi: 10.1016/j.freeradbiomed.2016.10.137 97 Curcumin Attenuates S-Glutathionylation of the NLRP3 Protein and Alters NLRP3 Inflammasome Protein-Protein Interactions in LPS/CNC-AEMA2 Stimulated-Macrophages Mostafa Elbery1, Adham Sabra1, Andrew Guglielmo1, Milena Mattes Cerveira1, Fernanda Ghenov1, Rajesh Sunasee1, and Karina Ckless1 1 SUNY Plattsburgh, USA Recently we have demonstrated that needle-like, cellulose-based nanomaterials, such as cationic derivatives of cellulose nanocrystals (CNCs, CNC-AEMA2) evoke immunological responses through NLRP3 inflammasome/IL-1β inflammatory pathway. We also previously demonstrated that curcumin, a naturally occurring polyphenolic compound isolated from Curcuma longa (Zingiberaceae), was able to suppress, at least in part, this immunological response, as observed by diminished IL-1β secretion in LPS/CNC-AEMA2-stimulated macrophages. These results are not a totally surprise because curcumin is a well-known antioxidant and antinflammatory natural compound. In addition to acting as “scavenger” of reactive oxygen species (ROS), several studies indicate that curcumin also upregulates antioxidant enzymes by activating the Nfr2 signaling pathway. However, the mechanisms by which this natural compound exerts its protective activity is still under investigation. We hypothesize that curcumin may also affect the redox status, such as S-glutathionylation, of key proteins involved in the NLRP3 inflammasome/IL-1β pathway, and therefore impacts their protein-protein interactions. The goal of this study was to investigate the effects of curcumin on the Sglutathionylation of NLRP3 induced by CNC-AEMA2 in LPSprimed mouse macrophages (J774A.1), as well as interactions among proteins of the NLRP3 inflammasome complex using immunoprecipitation and Western blot techniques. Cells were primed for 4h with lipopolysaccharide (LPS) in presence or absence of curcumin, and CNC-AEMA2 was added for another 20h. Our main finding indicates that the addition of curcumin concomitantly with LPS caused the greatest decrease in NLRP3 and increase in caspase-1 S-glutathionylation, which appears to favor protein-protein interactions in the NLRP3 complex. Taking together our results suggest that, at least in part, the antinflammatory activity of curcumin is associated with changes in S-glutathionylation of key NLRP3 inflammasome components, and perhaps resulting in sustained complex assembly and suppression of IL-1β secretion.
doi: 10.1016/j.freeradbiomed.2016.10.138
SfRBM / SFRRI 2016
S53
doi: 10.1016/j.freeradbiomed.2016.10.136 96 Monocytic Glutaredoxin 1 Protects Mice against Obesity, Hyperglycemia and Atherosclerosis Kevin Downs1, Sina Tavakoli1, John D. Short1, Huynh Nga Nguyen1, and Reto Asmis1 1 University of Texas Health Science Center at San Antonio, USA We previously reported that glutaredoxin 1 (Grx1) protects monocytes from metabolic-stress induced priming, dysregulation and hypersensitization to chemokines. To address the role of monocytic Grx1 in mice and in the development of atherogenesis and obesity, we transplanted bone marrow (BM) from either wildtype (WT) or Grx1-/- (Grx1Leuko -/-) donor mice into atherosclerosisprone LDLR-/- mice and fed these mice a high-fat diet (HFD) for up to 20 weeks. Grx1Leuko -/- mice showed accelerated weight gain after 9 weeks followed by early onset of hyperglycemia. After 6 weeks on HFD, atherosclerotic lesions were slightly larger in Grx1Leuko -/mice than in WT, but the differences did not reach statistical significance. However, after 20 weeks, Grx1Leuko -/- mice showed 36% larger lesions than WT-BM recipients, and monocyte chemotaxis in vivo was increased 1.6-fold. Furthermore, compared to WT-BM recipients, adipose tissues and livers of Grx1Leuko -/- mice also showed increased macrophage content and elevated tissue inflammation as determined by IHC and qRT-PCR. Adipose tissue in particular, showed significant increases in the expression of proinflammatory genes in addition to an increased abundance proinflammatory “crown-like” structures. Gene expression analysis by qRT-PCR of peritoneal macrophages revealed that macrophages isolated from Grx1-/- mice also have increased expression of pro-inflammatory M1-associated genes and decreased M2-associated genes after activation with INF+TNF and IL-4 respectively. Additionally, macrophages from Grx1-/- mice exposed to metabolic stress also display increased protein-Sglutathionylation, enhanced hypersensitization to chemokine, and impaired autophagy compared to wild-type cells. Taken together, our data show that loss of monocytic Grx1 worsens monocyte priming and accelerates the infiltration of dysfunctional monocyte-
derived macrophages into tissues, such as aorta, liver and adipose tissues. We conclude that monocytic Grx1 is critical for maintaining metabolic homeostasis in mice and protects against obesity and atherosclerosis.
doi: 10.1016/j.freeradbiomed.2016.10.137 97 Curcumin Attenuates S-Glutathionylation of the NLRP3 Protein and Alters NLRP3 Inflammasome Protein-Protein Interactions in LPS/CNC-AEMA2 Stimulated-Macrophages Mostafa Elbery1, Adham Sabra1, Andrew Guglielmo1, Milena Mattes Cerveira1, Fernanda Ghenov1, Rajesh Sunasee1, and Karina Ckless1 1 SUNY Plattsburgh, USA Recently we have demonstrated that needle-like, cellulose-based nanomaterials, such as cationic derivatives of cellulose nanocrystals (CNCs, CNC-AEMA2) evoke immunological responses through NLRP3 inflammasome/IL-1β inflammatory pathway. We also previously demonstrated that curcumin, a naturally occurring polyphenolic compound isolated from Curcuma longa (Zingiberaceae), was able to suppress, at least in part, this immunological response, as observed by diminished IL-1β secretion in LPS/CNC-AEMA2-stimulated macrophages. These results are not a totally surprise because curcumin is a well-known antioxidant and antinflammatory natural compound. In addition to acting as “scavenger” of reactive oxygen species (ROS), several studies indicate that curcumin also upregulates antioxidant enzymes by activating the Nfr2 signaling pathway. However, the mechanisms by which this natural compound exerts its protective activity is still under investigation. We hypothesize that curcumin may also affect the redox status, such as S-glutathionylation, of key proteins involved in the NLRP3 inflammasome/IL-1β pathway, and therefore impacts their protein-protein interactions. The goal of this study was to investigate the effects of curcumin on the Sglutathionylation of NLRP3 induced by CNC-AEMA2 in LPSprimed mouse macrophages (J774A.1), as well as interactions among proteins of the NLRP3 inflammasome complex using immunoprecipitation and Western blot techniques. Cells were primed for 4h with lipopolysaccharide (LPS) in presence or absence of curcumin, and CNC-AEMA2 was added for another 20h. Our main finding indicates that the addition of curcumin concomitantly with LPS caused the greatest decrease in NLRP3 and increase in caspase-1 S-glutathionylation, which appears to favor protein-protein interactions in the NLRP3 complex. Taking together our results suggest that, at least in part, the antinflammatory activity of curcumin is associated with changes in S-glutathionylation of key NLRP3 inflammasome components, and perhaps resulting in sustained complex assembly and suppression of IL-1β secretion.
doi: 10.1016/j.freeradbiomed.2016.10.138
SfRBM / SFRRI 2016
S53