ABSTRACT
Gut eubiosis is essential for the host's health. In athletes, the gut microbiota can be altered by several factors, including diets. While eubiotic gut microbiota in elite rugby players has been reported, our survey found that university rugby players suffered from loose stools and frequent urgency to defecate. To establish the causes of the condition, the microbiota and the concentrations of organic acids in fecal samples of university male rugby players (URP) were analyzed and compared with those of age-matching, non-rugby playing males (control). Body mass indices were significantly (p < 0.05) different between groups. Chao1 index was significant (p < 0.05) lower in URP than in control. The relative abundances of phyla Firmicutes and Bacteroidetes were significantly (p < 0.05) higher and lower, respectively, in URP than in control. Potential pathobiont genera Collinsella, Enterobacter, and Haemophilus were significantly (p < 0.05) abundant, whereas beneficial Akkermansia was lower (p < 0.05) in URP than in control. Succinate, a potential causative of gut inflammation, was five-fold higher in URP than in controls. Our findings all but confirmed that the dysbiotic status of gut in URP.
ABSTRACT
The relative abundance of phosphoinositide (PI) species on the phagosome membrane fluctuates over the course of phagocytosis. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 rapidly increase in the forming of the phagocytic cup, following which they disappear after sealing of the cup. In the present study, we monitored the clearance of these PI species using the enhanced green fluorescent protein-fused pleckstrin homology domain of Akt, a fluorescence probe that binds both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in Raw 264.7 macrophages. The clearance of PIs was much faster when the phagocytosed particles were coated with IgG. The effect of IgG was not observed in the macrophages deficient in FcγRIIb, an inhibitory IgG receptor. To identify the lipid phosphatases responsible for the FcγRIIb-accelerated PI clearance, we prepared a panel of lipid phosphatase-deficient cells. The lack of a PI 5-phosphatase Src homology 2 domain-containing inositol-5-phosphatase (SHIP)1 or SHIP2 impaired the FcγRIIb-accelerated clearance of PIs. The lack of a PI 4-phosphatase Inpp4a also impaired the accelerated PIs clearance. In the FcγRIIb- and Inpp4a-deficient cells, acidification of the formed phagosome was slowed. These results suggested that FcγRIIb drives the sequential dephosphorylation system comprising SHIPs and Inpp4a, and accelerates phagosome acidification.
Subject(s)
Macrophages/metabolism , Oncogene Protein v-akt/metabolism , Phagocytosis , Phagosomes/metabolism , Phosphoric Monoester Hydrolases/metabolism , Receptors, IgG/metabolism , Animals , Hydrogen-Ion Concentration , Immunoglobulin G/metabolism , Macrophages/immunology , Mice , Oncogene Protein v-akt/genetics , Phosphatidylinositol Phosphates/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/genetics , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphorylation , Protein Binding , RAW 264.7 Cells , RNA, Small Interfering/genetics , Receptors, IgG/geneticsABSTRACT
Phosphoinositide 5'-phosphatases have been implicated in the regulation of phagocytosis. However, their precise roles in the phagocytic process are poorly understood. We prepared RAW264.7 macrophages deficient in Inpp5e (shInpp5e) to clarify the role of this lipid phosphatase. In the shInpp5e cells, the uptake of solid particles was increased and the rate of phagosome acidification was accelerated. As expected, levels of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 were increased and decreased respectively, on the forming phagocytic cups of these cells. Unexpectedly, the most prominent consequence of the Inpp5e deficiency was the decreased accumulation of PtdIns3P and Rab5 on the phagosome. The expression of a constitutively active form of Rab5b in the shInpp5e cells rescued the PtdIns3P accumulation. Rab20 has been reported to regulate the activity of Rabex5, a guanine nucleotide exchange factor for Rab5. The association of Rab20 with the phagosome was remarkably abrogated in the shInpp5e cells. Over-expression of Rab20 increased phagosomal PtdIns3P accumulation and delayed its elimination. These results suggest that Inpp5e, through functional interactions with Rab20 on the phagosome, activates Rab5, which, in turn, increases PtdIns3P and delays phagosome acidification.
Subject(s)
Phagosomes/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphoric Monoester Hydrolases/physiology , rab GTP-Binding Proteins/metabolism , rab5 GTP-Binding Proteins/metabolism , Acids/metabolism , Animals , Cells, Cultured , Macrophages/metabolism , Mice , Phagocytosis/genetics , Protein Binding , TransfectionABSTRACT
PtdIns(3)P (phosphatidylinositol 3-phosphate) is a signaling molecule important for phagosome maturation. The major role of Vps34 in production of phagosomal PtdIns(3)P has been indicated. However, the fate of the newly generated PtdIns(3)P has not been well described. Here we show that elimination of PtdIns(3)P from phagosomal membrane was significantly delayed in RAW264.7 macrophages lacking PTEN or PIKfyve. In the PTEN-deficient cells treated with a PIKfyve inhibitor, degradation of PtdIns(3)P was almost lost, indicating that PTEN and PIKfyve are two major players in phagosomal PtdIns(3)P metabolism.
