Int J Stem Cells.  2019 Nov;12(3):419-429. 10.15283/ijsc19115.

Perivascular Stem Cells Suppress Inflammasome Activation during Inflammatory Responses in Macrophages

• Kim J
• Kim WJ
• Ha KS
• Han ET
• Park WS
• Yang SR
• Hong SH

• KMID: 2465882
• DOI: http://doi.org/10.15283/ijsc19115

Abstract

BACKGROUND AND OBJECTIVES
Perivascular stem cells (PVCs) have been identified as precursors of mesenchymal stem cells (MSCs) that offer promising prospects for application in the development of cellular therapies. Although PVCs have been demonstrated to have greater therapeutic potential compared to bone marrow and adipose tissue-derived MSCs in various diseases, the regulatory role of PVCs on inflammasome activation during macrophage-mediated inflammatory responses has not been investigated.
METHODS AND RESULTS
In this study, we found that the PVC secretome effectively alleviates secretion of both caspase-1 and interleukin-1β in lipopolysaccharide-primed and activated human and murine macrophages by blocking inflammasome activation and attenuating the production of mitochondrial reactive oxygen species (ROS). We further showed that the PVC secretome significantly reduces inflammatory responses and endoplasmic reticulum stress in peritoneal macrophages in a mouse model of monosodium urate-induced peritonitis. A cytokine antibody array analysis revealed that the PVC secretome contains high levels of serpin E1 and angiogenin, which may be responsible for the inhibitory effects on mitochondrial ROS generation as well as on inflammasome activation.
CONCLUSIONS
Our results suggest that PVCs may be therapeutically useful for the treatment of macrophage- and inflammation-mediated diseases by paracrine action via the secretion of various biological factors.

Keyword

Perivascular stem cells; Inflammation; Inflammasome; Macrophage

MeSH Terms

Animals
Biological Factors
Bone Marrow
Endoplasmic Reticulum Stress
Humans
Inflammasomes*
Inflammation
Macrophages*
Macrophages, Peritoneal
Mesenchymal Stromal Cells
Mice
Peritonitis
Plasminogen Activator Inhibitor 1
Reactive Oxygen Species
Stem Cells*
Biological Factors
Inflammasomes
Plasminogen Activator Inhibitor 1
Reactive Oxygen Species

Figure

• 

  Fig. 1 The inhibitory effect of PVC-CM on inflammasome activation in human macrophage. PMA-differentiated THP-1 cells were primed with LPS (1 μg/ml) in RPMI medium containing 10% FBS and antibiotics for 3 h, followed by incubation with inflammasome activators for 1 h. (A) Schematic diagram of steps involved in the process of inflammasome activation. (B) Cells were cultured in RPMI media containing the indicated concentration of PVC-CM or ATP (2 mM) as a positive control for 1 h. IL-1β secretion was analyzed by immunoblotting. (C) PMA-differentiated THP-1 cells were treated with ATP (2 mM), flagellin (0.5 μg/ml), or dsDNA (2 μg/ml) for 1 h in the presence of PVC-CM. Cellular supernatant (Sup), lysate (Lys) and cross-linked pellets (Pellet) from whole-cell lysates were analyzed with the indicated anti-sera in an immunoblot assay. (D, E) Secreted caspase-1 and IL-1β were quantitated by an ELISA-based assay kit and the data are presented as bar graphs. Error bars indicate SD (*p<0.05, **p<0.01, ***p<0.001).

• 

  Fig. 2 PVC-CM inhibits the inflammasomes activation and caspase-1 activity in mouse peritoneal macrophage. Peritoneal macrophages were isolated, primed with LPS for 3 h, and subjected to the indicated inflammasome triggers in the present of PVC-CM. (A) Sup, Lys, and Pellet from whole-cell lysates were analyzed for caspase-1, IL-1β, or ASC pyroptosome by immunoblot assay. (B) Supernatant mouse IL-1β levels were measured by ELISA. (C) rhCasp1 was incubated with its substrate (YVAD-pNA) in the presence of PVC-CM as indicated. Relative Casp1 activity in the reaction without the rhCasp1 was set as 0% and that in the reaction with Casp1 and YVAD-pNA without PVC-CM was set as 100%. Z-VAD indicates Z-VAD-FMK, a pan-caspase inhibitor. (D) The cytotoxicity of PVC-CM was measured after applying the indicated dosage of PVC-CM to PMA-differentiated THP-1 for 1 h, identical with the inflammasome-activating step. The survival rate of the Triton-treated group was set as 0% and that of the non-treated group was set as 100%. Error bars indicate SD (*p<0.05, **p<0.01, ***p<0.001).

• 

  Fig. 3 PVC-CM reduces inflammation and ER stress in in macrophages of MSU-induced peritonitis. (A) Schematic diagram of experiments using the peritonitis mouse model (n=8 per group). (B) PECs were determined by counting exudate cells with a cell counter. (C) Cytospins of isolated PECs were stained with Wright-Giemsa stain. (D, E) Relative mRNA levels of inflammatory (IL-1α, IL-1β, NLRP3, and Casp1) and ER stress-related genes (ATF6, PERK, elk2α, and IRE-1) were analyzed using SYBR green-based quantitative real-time PCR. Error bars indicate SD (*p<0.05, **p<0.01).

• 

  Fig. 4 Secretome analysis. Proteome profiler arrays were evaluated as described in Methods. (A) The heat map represents the subsequent image analysis and quantification of pixel intensity for each spot. (B, C) LPS-primed THP-1 cells treated with inflammasome triggers in the absence or presence of rhSerpin E1 or rhAngiogenin for 1 h. IL-1β levels in the supernatants were measured by ELISA. (D) Caspase-1 activity was determined by colorimetric assay. Error bars indicate SD (*p<0.05, **p<0.01, ***p<0.001).

• 

  Fig. 5 The Mechanism underlying the inhibitory effect of PVC-CM on NLRP3 inflammasome via mitochondrial ROS generation. (A) LPS-primed THP-1 cells treated with ATP (2 mM) and the indicated DPI (100 to 10 μM) for 1 h. Sup and Lys were analyzed by immunoblot using the indicated anti-sera. (B) LPS-primed THP-1 cells were treated with rotenone (20 μM) and the indicated dosages of PVC-CM for 6 h, after which mitochondrial ROS generation was analyzed by immunoblot. (C) Mitochondrial ROS (MitoSOX) were quantified using a fluorometric microplate reader. (D) Relative fluorescence intensities of Mito Tracker (mitochondria) and MitoSOX (mitochondrial ROS) were analyzed using confocal microscopy. Hoechst was used for nuclear staining. Error bars indicate SD (*p<0.05, **p<0.01, ***p<0.001).

Reference

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