8 research outputs found
Roles of Macrophage Exosomes in Immune Response to Calcium Oxalate Monohydrate Crystals
In kidney stone disease, macrophages secrete various mediators via classical secretory pathway and cause renal interstitial inflammation. However, whether their extracellular vesicles, particularly exosomes, are involved in kidney stone pathogenesis remained unknown. This study investigated alterations in exosomal proteome of U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) after exposure to calcium oxalate monohydrate (COM) crystals for 16-h using 2-DE-based proteomics approach. Six significantly altered proteins in COM-treated exosomes were successfully identified by nanoscale liquid chromatography–electrospray ionization–electron transfer dissociation tandem mass spectrometry as proteins involved mainly in immune processes, including T-cell activation and homeostasis, Fcγ receptor-mediated phagocytosis, interferon-γ (IFN-γ) regulation, and cell migration/movement. The decreased heat shock protein 90-beta (HSP90β) and increased vimentin were confirmed by Western blotting. ELISA showed that the COM-treated macrophages produced greater level of interleukin-1β (IL-1β), one of the markers for inflammasome activation. Functional studies demonstrated that COM-treated exosomes enhanced monocyte and T-cell migration, monocyte activation and macrophage phagocytic activity, but on the other hand, reduced T-cell activation. In addition, COM-treated exosomes enhanced production of proinflammatory cytokine IL-8 by monocytes that could be restored to its basal level by small-interfering RNA targeting on vimentin (si-Vimentin). Moreover, si-Vimentin could also abolish effects of COM-treated exosomes on monocyte and T-cell migration as well as macrophage phagocytic activity. These findings provided some implications to the immune response during kidney stone pathogenesis via exosomal pathway of macrophages after exposure to COM crystals
Alterations in Macrophage Cellular Proteome Induced by Calcium Oxalate Crystals: The Association of HSP90 and F‑Actin Is Important for Phagosome Formation
The
presence of macrophages in renal interstitium is the key feature
of progressive renal inflammation in kidney stone disease. However,
response of macrophages to calcium oxalate monohydrate (COM) crystals,
the major crystalline composition of kidney stone, remained unclear.
This study aimed to investigate alterations in the cellular proteome
of macrophages induced by COM crystals using a proteomics approach.
U937-derived macrophages (by phorbol-12-myristate-13-acetate activation)
were incubated without or with 100 μg/mL COM crystals for 24
h. Their cellular proteins were resolved by 2-DE (<i>n</i> = 10 gels; 5 were derived from 5 independent cultures in each group)
and visualized with Deep Purple fluorescent dye. Spot matching, quantitative
intensity analysis, and statistics revealed 18 differentially expressed
protein spots, which were successfully identified by Q-TOF MS and
MS/MS analyses. The altered levels of α-tubulin, β-actin
and ezrin were validated by Western blot analysis. Protein interaction
network analysis using STRING software showed that 90 kDa heat shock
protein (HSP90) was associated with β-actin and α-tubulin
(all these three proteins were increased in the COM-treated macrophages).
Multiple immunofluorescence stainings confirmed the associations of
HSP90 with filamentous form of actin (F-actin) and α-tubulin.
However, only the association between HSP90 and F-actin was found
on the phagosome membrane surrounding COM crystal, indicating that
the association of HSP90 with F-actin, but not with α-tubulin,
is important for phagosome formation. Silencing of HSP90 (siHSP90)
reduced expression of cytoskeletal proteins and phagosome marker (Rab5)
and successfully diminished COM crystal-induced phagocytosis and migration
of macrophages. Our findings enlightened the significant role of these
altered proteins, especially HSP90, in enhanced phagocytic activity
of the COM-exposed macrophages
Alterations in Macrophage Cellular Proteome Induced by Calcium Oxalate Crystals: The Association of HSP90 and F‑Actin Is Important for Phagosome Formation
The
presence of macrophages in renal interstitium is the key feature
of progressive renal inflammation in kidney stone disease. However,
response of macrophages to calcium oxalate monohydrate (COM) crystals,
the major crystalline composition of kidney stone, remained unclear.
This study aimed to investigate alterations in the cellular proteome
of macrophages induced by COM crystals using a proteomics approach.
U937-derived macrophages (by phorbol-12-myristate-13-acetate activation)
were incubated without or with 100 μg/mL COM crystals for 24
h. Their cellular proteins were resolved by 2-DE (<i>n</i> = 10 gels; 5 were derived from 5 independent cultures in each group)
and visualized with Deep Purple fluorescent dye. Spot matching, quantitative
intensity analysis, and statistics revealed 18 differentially expressed
protein spots, which were successfully identified by Q-TOF MS and
MS/MS analyses. The altered levels of α-tubulin, β-actin
and ezrin were validated by Western blot analysis. Protein interaction
network analysis using STRING software showed that 90 kDa heat shock
protein (HSP90) was associated with β-actin and α-tubulin
(all these three proteins were increased in the COM-treated macrophages).
Multiple immunofluorescence stainings confirmed the associations of
HSP90 with filamentous form of actin (F-actin) and α-tubulin.
However, only the association between HSP90 and F-actin was found
on the phagosome membrane surrounding COM crystal, indicating that
the association of HSP90 with F-actin, but not with α-tubulin,
is important for phagosome formation. Silencing of HSP90 (siHSP90)
reduced expression of cytoskeletal proteins and phagosome marker (Rab5)
and successfully diminished COM crystal-induced phagocytosis and migration
of macrophages. Our findings enlightened the significant role of these
altered proteins, especially HSP90, in enhanced phagocytic activity
of the COM-exposed macrophages
Image_1.PDF
<p>In kidney stone disease, macrophages secrete various mediators via classical secretory pathway and cause renal interstitial inflammation. However, whether their extracellular vesicles, particularly exosomes, are involved in kidney stone pathogenesis remained unknown. This study investigated alterations in exosomal proteome of U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) after exposure to calcium oxalate monohydrate (COM) crystals for 16-h using 2-DE-based proteomics approach. Six significantly altered proteins in COM-treated exosomes were successfully identified by nanoscale liquid chromatography–electrospray ionization–electron transfer dissociation tandem mass spectrometry as proteins involved mainly in immune processes, including T-cell activation and homeostasis, Fcγ receptor-mediated phagocytosis, interferon-γ (IFN-γ) regulation, and cell migration/movement. The decreased heat shock protein 90-beta (HSP90β) and increased vimentin were confirmed by Western blotting. ELISA showed that the COM-treated macrophages produced greater level of interleukin-1β (IL-1β), one of the markers for inflammasome activation. Functional studies demonstrated that COM-treated exosomes enhanced monocyte and T-cell migration, monocyte activation and macrophage phagocytic activity, but on the other hand, reduced T-cell activation. In addition, COM-treated exosomes enhanced production of proinflammatory cytokine IL-8 by monocytes that could be restored to its basal level by small-interfering RNA targeting on vimentin (si-Vimentin). Moreover, si-Vimentin could also abolish effects of COM-treated exosomes on monocyte and T-cell migration as well as macrophage phagocytic activity. These findings provided some implications to the immune response during kidney stone pathogenesis via exosomal pathway of macrophages after exposure to COM crystals.</p
Chromosome-centric Human Proteome Project (C-HPP): Chromosome 12
Following an official announcement of the Chromosome-centric Human Proteome Project (C-HPP), the Chromosome 12 (Ch12) Consortium has been established by five representative teams from five Asian countries including Thailand (Siriraj Hospital, Mahidol University), Singapore (National University of Singapore), Taiwan (Academia Sinica), Hong Kong (The Chinese University of Hong Kong), and India (Institute of Bioinformatics). We have worked closely together to extensively and systematically analyze all missing and known proteins encoded by Ch12 for their tissue/cellular/subcellular localizations. The target organs/tissues/cells include kidney, brain, gastrointestinal tissues, blood/immune cells, and stem cells. In the later phase, post-translational modifications and functional significance of Ch12-encoded proteins as well as their associations with human diseases (i.e., immune diseases, metabolic disorders, and cancers) will be defined. We have collaborated with other chromosome teams, Human Kidney and Urine Proteome Project (HKUPP), AOHUPO Membrane Proteomics Initiative, and other existing HUPO initiatives in the Biology/Disease-Based Human Proteome Project (B/D-HPP) to delineate functional roles and medical implications of Ch12-encoded proteins. The data set to be obtained from this multicountry consortium will be an important piece of the jigsaw puzzle to fulfill the missions and goals of the C-HPP and the global Human Proteome Project (HPP)
Chromosome-centric Human Proteome Project (C-HPP): Chromosome 12
Following an official announcement
of the Chromosome-centric Human
Proteome Project (C-HPP), the Chromosome 12 (Ch12) Consortium has
been established by five representative teams from five Asian countries
including Thailand (Siriraj Hospital, Mahidol University), Singapore
(National University of Singapore), Taiwan (Academia Sinica), Hong
Kong (The Chinese University of Hong Kong), and India (Institute of
Bioinformatics). We have worked closely together to extensively and
systematically analyze all missing and known proteins encoded by Ch12
for their tissue/cellular/subcellular localizations. The target organs/tissues/cells
include kidney, brain, gastrointestinal tissues, blood/immune cells,
and stem cells. In the later phase, post-translational modifications
and functional significance of Ch12-encoded proteins as well as their
associations with human diseases (i.e., immune diseases, metabolic
disorders, and cancers) will be defined. We have collaborated with
other chromosome teams, Human Kidney and Urine Proteome Project (HKUPP),
AOHUPO Membrane Proteomics Initiative, and other existing HUPO initiatives
in the Biology/Disease-Based Human Proteome Project (B/D-HPP) to delineate
functional roles and medical implications of Ch12-encoded proteins.
The data set to be obtained from this multicountry consortium will
be an important piece of the jigsaw puzzle to fulfill the missions
and goals of the C-HPP and the global Human Proteome Project (HPP)