The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

Activation of the NOD-like receptor protein 3 (NLRP3) inflammasome complex causes the processing and release of mature IL-1β, with mitochondria playing key roles in its assembly. An orally active NLRP3 inflammasome inhibitor would be a significant advance in therapy for IL-1β-driven diseases. To overcome both, the variability among primary immune cells and the limitations of genetic manipulation of differentiated human or murine macrophages, we developed a simplified, reliable and relevant cell-based model for studying the NLRP3 inflammasome using the undifferentiated human myelomonocytic cell line THP1. We established that undifferentiated THP1 cells are fully competent for activation of the NLRP3 inflammasome and production of IL-1β, without differentiation into macrophages. CP-456,773 is a potent and selective inhibitor of the NLRP3 inflammasome, and it is an analogue of glyburide, a sulfonylurea receptor (SUR) inhibitor. Despite the extensive experimental use of CP-456,773, its molecular target remains unknown. Here we tested the hypothesis that mitochondrial ABCb7 or ABCb10 could be the pharmacologic targets of CP-456,773. We optimized a viral shRNA transduction method for genetic manipulations in THP1 cells and generated ABCb7 and ABCb10 knockdown (KD) THP1 cells. We demonstrate that NLRP3 inflammasome activation and CP-456,773 pharmacology are not altered in ABCb7- or ABCb10-deficient THP1 cells. For ABCb10, we confirmed these results using CRISPR/CAS9-mediated ABCb10 knockout (KO) THP1 sub-lines. In studies of mitochondrial fitness, we found that a previously observed reduction in oxygen consumption rate (OCR) following nigericin treatment was completely blocked in NLRP3 KO cells. Our data demonstrating that CP-456,773 rescues the NLRP3-dependent nigericin-induced decline in OCR and protects undifferentiated THP1 cells from nigericin-induced pyroptosis are consistent with the possibility that the NLRP3 protein itself may be the molecular target of CP-456,773. Moreover, we showed that ABCb10 KO THP1 cells exhibit increased rates of basal ATP production and glycolysis, suggesting an important role for ABCb10 in mitochondrial metabolism. Finally, RNA-Seq analysis of ABCb7 and ABCb10 KD in undifferentiated THP1 cells indicate new functions for these proteins, including cell communication and migration, apoptosis and cell adhesion. Overall, our findings demonstrate that undifferentiated THP1 cells are an ideal system in which to study the NLRP3 inflammasome.

Bakgrund: Användningen av nanopartiklar ökar varje dag men mycket är oklart i vilkenutsträckning nanopartiklar har hälsopåverkan hos människor. I studier har det visats attnanopartiklar under 100 nm kan passera cellmembranet och under 40 nm kan passeranuklearmembran. I dagsläget används guldnanopartiklar (GNP) inom medicin för visualisering, diagnostisering och läkemedelsbärare. En av biverkningar som GNP kan ha är inflammation. Det finns flera studier som försökt redogöra GNP:s effekter samt hur de uppkommer men det är intehelt klarlagt. Vidare är det inte helt tydligt vilka egenskaper hos GNP som står för de olikaeffekterna. Syfte: Syftet med studien är att undersöka rollen som GNP:s storlekar och former har i NLRfamily pyrin domain containing 3 (NLRP3) inflammasomens interleukin-1β-frisättning hoshuman leukemia monocytic cell line (THP1-celler). Metod: Studien är en experimentell studie där THP1-celler differentieras och exponeras för GNPi olika storlekar, former och koncentrationer. LUMIT immunoassay används sedan för att erhållade olika interleukin-1β-koncentrationerna (IL-1β). Resultat: I datan som erhölls efter att experimenten utförts observerades det att GNP 30 nm i 40μg/mL gav störst inflammasominduktion med en IL-1β-koncentration på 645 ±388 pg/mL.Nästhögst IL-1β-koncentration gav GNP 100 nm i 40 μg/mL på 592 ±270 pg/mL. Slutsats: Nakna GNP kan inducera inflammasomaktivering och på så sättIL-1β-koncentrationshöjningar. De “gold urchins” som undersökts visade sig ha svagarepåverkan på cellerna jämfört med de sfäriska GNP.

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La plupart des pathologies, qu'elles soient d'origine génétique ou non trouvent leur origine dans l'altération structural et/ou fonctionnelle de protéines d'intérêt. L'agrégation, la glycation et l'oxydation constituent les principaux types de modification protéique fréquemment observées dans de nombreuses maladies tels que le diabète, l'athérosclérose ou encore les maladies neurodégénératives. Généralement, l'altération des activités biologiques de ces protéines est étroitement corrélée avec ses changements conformationnels et structuraux. L'albumine représente la protéine circulante la plus abondante et constitue le plus important antioxydant du plasma sanguin susceptible d'être exposée à un stress glycoxydant accru, et aboutir à la formation de Produits Avancés de Glycation (AGEs) dont les effets peuvent être néfastes au niveau cellulaire et tissulaire. Dans cette étude, je me suis intéressé à l'effet de trois types d'altérations, que sont l'agrégation, la glycation et l'oxydation, sur la structure, le statut redox et l'activité antioxydante de l'albumine ainsi que l'incidence de cette protéine modifiée sur la physiologie de différentes lignées cellulaires. Au cours de ce travail, j'ai abordé les quatre points suivants : 1. Afin de mieux comprendre l'impact de ces processus altératifs sur les propriétés antioxydantes de l'albumine, j'ai étudié, en utilisant des techniques de la Biochimie et de la Biophysique, les modifications structurales de la protéine soumise successivement à la glycation, l'agrégation et aux deux processus simultanément. 2. J'ai mis en évidence et identifié des cibles protéiques sensibles à l'oxydation au niveau d'adipocytes matures humains incubés en présence d'albumine bovine glycoxydée in vitro. 3. J'ai effectué une comparaison de l'action différentielle des modèles in vitro de d'AGEs préparés à partir de l'albumine d'origine bovine ou humaine au niveau de monocytes THP1. 4. Enfin, afin de valider les caractéristiques structurales et biologiques obtenues à partir de nos modèles in vitro d'AGEs dérivés de l'albumine, j'ai étudié sur des modèles purifiés à partir de plasmas de sujets, l'état structural, redox et antioxydant de l'albumine altérée in vivo. Les résultats obtenus lors de ce travail permettent de mettre en avant l'importance de la structure native de la plus abondante des protéines circulantes sur l'intégrité de ses fonctions biologiques et en particulier sur son activité antioxydante. Également, cette étude apporte un nouvel éclairage sur l'implication de l'albumine altérée dans de nombreux désordres métaboliques tels que le diabète.

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