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Study of the role of cytosolic phospholipase A2 alpha in eicosanoid generation and thymocyte maturation in the thymus.

Rousseau M, Naika GS, Perron J, Jacques F, Gelb MH, Boilard E - PLoS ONE (2015)

Bottom Line: The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses.As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations.The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Faculté de Médecine de l'Université Laval, Québec, QC, Canada.

ABSTRACT
The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA2α.

No MeSH data available.


Related in: MedlinePlus

High concentration of PP impacts thymocyte maturation independently of cPLA2α inhibition.A. Representative distribution of the thymocyte subpopulations in WT and KO cPLA2α FTOCs after 5 days of culture in absence or presence of 1μM of PP. Thymocytes were identified with fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8 and analyzed by flow cytometry. B. WT and KO cPLA2α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of 1μM of PP. After mechanical dissociation of fetal thymuses, thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 4 to 9 independent experiments and the number of fetal thymuses for each condition is: cPLA2+/+ and Diluent (n = 22); cPLA2+/+ and 1μM PP (n = 13); cPLA2-/- and diluent (n = 9); cPLA2-/- and 1μM PP (n = 6). * P< .05; ** P< .01; *** P< .001.
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pone.0126204.g005: High concentration of PP impacts thymocyte maturation independently of cPLA2α inhibition.A. Representative distribution of the thymocyte subpopulations in WT and KO cPLA2α FTOCs after 5 days of culture in absence or presence of 1μM of PP. Thymocytes were identified with fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8 and analyzed by flow cytometry. B. WT and KO cPLA2α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of 1μM of PP. After mechanical dissociation of fetal thymuses, thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 4 to 9 independent experiments and the number of fetal thymuses for each condition is: cPLA2+/+ and Diluent (n = 22); cPLA2+/+ and 1μM PP (n = 13); cPLA2-/- and diluent (n = 9); cPLA2-/- and 1μM PP (n = 6). * P< .05; ** P< .01; *** P< .001.

Mentions: We next used a pharmacological approach to confirm our observations made in genetically engineered mice. The cPLA2α inhibitor pyrrophenone (PP) [48] suppresses AA release from an activated monocytic cell line and PGE2 release by renal mesangial cells with an IC50 of 24nM and 8nM, respectively [48]. WT FTOCs were cultured during 5 days in absence or in presence of 10 and 100 nm of PP and the repartitioned thymocyte subpopulations were analyzed by flow cytometry. As for the genetic approach, cPLA2α appeared dispensable, as no differences were observed in the maturation of thymocytes in presence of PP compared to those left untreated (Fig 3A and 3B). Rocca et al. and Xu et al. observed that the culture to FTOCs in presence of high concentrations (40μM) of the COX-2 inhibitor NS-398 led to the blockade of thymocyte differentiation [21, 35]. This effect of NS-398 was considered unspecific, as it was recapitulated in COX-2 deficient FTOCs and it was not reversed by the addition of PGE2 [35]. Using high concentrations (1μM) of the cPLA2α inhibitor, we observed an increase and a decrease of DN and DP thymocyte populations, respectively (Fig 4A and 4B). Furthermore, we observed an increase of the two SP populations (Fig 4A and 4B). Thus, high dose of PP affects the maturation of mouse thymocytes. We next wished to confirm the specificity of the inhibitor, here using cPLA2α KO FTOCs. We observed that PP, used at 1 μM, impedes the maturation of thymocytes deficient in cPLA2α (Fig 5A and 5B). Furthermore, the exogenous addition of AA and of PGE2, which was reported involved in thymocyte maturation [21], to PP-treated FTOCs did not restore normal thymocyte maturation (Fig 6A and 6B). Taken together, these results demonstrate that high doses of PP inhibit thymocyte maturation through the inhibition of another target than cPLA2α, most likely irrelevant to AA and prostaglandin release.


Study of the role of cytosolic phospholipase A2 alpha in eicosanoid generation and thymocyte maturation in the thymus.

Rousseau M, Naika GS, Perron J, Jacques F, Gelb MH, Boilard E - PLoS ONE (2015)

High concentration of PP impacts thymocyte maturation independently of cPLA2α inhibition.A. Representative distribution of the thymocyte subpopulations in WT and KO cPLA2α FTOCs after 5 days of culture in absence or presence of 1μM of PP. Thymocytes were identified with fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8 and analyzed by flow cytometry. B. WT and KO cPLA2α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of 1μM of PP. After mechanical dissociation of fetal thymuses, thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 4 to 9 independent experiments and the number of fetal thymuses for each condition is: cPLA2+/+ and Diluent (n = 22); cPLA2+/+ and 1μM PP (n = 13); cPLA2-/- and diluent (n = 9); cPLA2-/- and 1μM PP (n = 6). * P< .05; ** P< .01; *** P< .001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4430275&req=5

pone.0126204.g005: High concentration of PP impacts thymocyte maturation independently of cPLA2α inhibition.A. Representative distribution of the thymocyte subpopulations in WT and KO cPLA2α FTOCs after 5 days of culture in absence or presence of 1μM of PP. Thymocytes were identified with fluorochrome-conjugated antibodies directed against CD3, CD4 and CD8 and analyzed by flow cytometry. B. WT and KO cPLA2α fetal thymuses were cultured during 5 days as FTOCs in absence or presence of 1μM of PP. After mechanical dissociation of fetal thymuses, thymocytes were labeled with fluorochrome-conjugated antibodies directed against CD3, CD4, and CD8 and analyzed by flow cytometry. Data are mean ± SEM of 4 to 9 independent experiments and the number of fetal thymuses for each condition is: cPLA2+/+ and Diluent (n = 22); cPLA2+/+ and 1μM PP (n = 13); cPLA2-/- and diluent (n = 9); cPLA2-/- and 1μM PP (n = 6). * P< .05; ** P< .01; *** P< .001.
Mentions: We next used a pharmacological approach to confirm our observations made in genetically engineered mice. The cPLA2α inhibitor pyrrophenone (PP) [48] suppresses AA release from an activated monocytic cell line and PGE2 release by renal mesangial cells with an IC50 of 24nM and 8nM, respectively [48]. WT FTOCs were cultured during 5 days in absence or in presence of 10 and 100 nm of PP and the repartitioned thymocyte subpopulations were analyzed by flow cytometry. As for the genetic approach, cPLA2α appeared dispensable, as no differences were observed in the maturation of thymocytes in presence of PP compared to those left untreated (Fig 3A and 3B). Rocca et al. and Xu et al. observed that the culture to FTOCs in presence of high concentrations (40μM) of the COX-2 inhibitor NS-398 led to the blockade of thymocyte differentiation [21, 35]. This effect of NS-398 was considered unspecific, as it was recapitulated in COX-2 deficient FTOCs and it was not reversed by the addition of PGE2 [35]. Using high concentrations (1μM) of the cPLA2α inhibitor, we observed an increase and a decrease of DN and DP thymocyte populations, respectively (Fig 4A and 4B). Furthermore, we observed an increase of the two SP populations (Fig 4A and 4B). Thus, high dose of PP affects the maturation of mouse thymocytes. We next wished to confirm the specificity of the inhibitor, here using cPLA2α KO FTOCs. We observed that PP, used at 1 μM, impedes the maturation of thymocytes deficient in cPLA2α (Fig 5A and 5B). Furthermore, the exogenous addition of AA and of PGE2, which was reported involved in thymocyte maturation [21], to PP-treated FTOCs did not restore normal thymocyte maturation (Fig 6A and 6B). Taken together, these results demonstrate that high doses of PP inhibit thymocyte maturation through the inhibition of another target than cPLA2α, most likely irrelevant to AA and prostaglandin release.

Bottom Line: The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses.As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations.The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Faculté de Médecine de l'Université Laval, Québec, QC, Canada.

ABSTRACT
The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA2α.

No MeSH data available.


Related in: MedlinePlus