Oxid Med Cell Longev. 2013;2013:186795. doi: 10.1155/2013/186795. Epub 2013 Apr 18.

Adaptive Redox Response of Mesenchymal Stromal Cells to Stimulation with Lipopolysaccharide Inflammagen: Mechanisms of Remodeling of Tissue Barriers in Sepsis

Nikolai V. Gorbunov, Bradley R. Garrison, Dennis P. McDaniel, Min Zhai, Pei-Jyun Liao, Dilber Nurmemet, and Juliann G. Kiang

Radiation Combined Injury Program, Armed Forces Radiobiology Research Institute, Bethesda, MD 20889-1402, USA. nikolaiv.gorbunov@gmail.com

 

ABSTRACT

Acute bacterial inflammation is accompanied by excessive production of reactive oxygen and nitrogen species (ROS and RNS), which ultimately results in redox-stress, a leading pathogenic factor of the septic multiple organ dysfunction syndromes. According to the current paradigm, the inflammatory redox-stress is primarily attributed to the defense responses of the reticuloendothelial, endothelial, and lymphoepithelial components of tissue barriers to infections. Meanwhile, a large body of data accumulated in the last decade has pointed to an emerging role of ubiquitous mesenchymal stromal cells (MSCs) playing in the antibacterial and inflammatory events. In conjunction with this evidence, investigation of cellular pathways up-regulated in MSCs under redox stress conditions may provide new insights into mechanisms driving homeostatic responses of defense barriers to infections. This report presents results of in vitro investigations of the redox response of mouse MSCs to stimulation with Lipopolysaccharide (LPS) inflammagen. We have shown that MSCs treated with LPS experienced redox-stress due to induction of nitric oxide synthase (iNOS) and release of RNS and ROS. The compensatory response of MSCs to the LPS-induced cytotoxic stress was associated with activation of a number of the adaptive redox-response elements such as NFkB, Ref1, TRX1, Nrf2 and HO1, and autophagy, a cellular homeostatic process of remodeling and turnover of compromised cellular constituents. We propose that the cell survival mechanisms activated in LPS-treated MSCs in vitro could be a part of adaptive responses employed by stromal cells under septic conditions.

PMID: 23710283

 

SUPPLEMENTARY

The objective of the study reported by Gorbunov et al. was to demonstrate coherent interaction between inflammatory oxidative stress and adaptive stress-response mechanisms in mesenchymal stromal cells (MSCs) challenged with the LPS inflammagen. The events occurred in the challenged MSCs were shown to be tightly associated with remodeling occurred in mitochondrial network, namely increase in production of the reactive oxygen species (ROS), formation of aberrant mitochondria, mitochondrial fission, and mitophagy. This is the first communication, which associates mitochondrial network with the response of mesenchymal stromal cells to inflammagenes. The reported observations are in concord with a new emerging paradigm, which proposes mitochondria-based innate defense platform for execution of intracellular antibacterial/inflammatory signalling mediated by TLRs/PAMPs/DAMPs and IRGM networks [1,2,3].

MSCs challenged with LPS expressed numerous inflammatory cytokines and iNOS. Expression of these effectors occurred in NFκB – dependent manner and suppressed by PDTC, an NFκB inhibitor; note the challenge caused a prompt (within 1 h) increase in the nuclear translocation of (p65) NFκB. A dramatic accumulation of iNOS protein (Fig. 1) was accompanied by excessive production of ROS and RNS with evident co-localization of oxidative stress with mitochondria (Fig. 2). Tentatively, these effects can cause protracted oxidative damage to the cell and therefore, are accompanied by up-regulation of redox defense mechanisms [4, 5]  In order to mitigate this “self-inflicted” oxidative damage, the challenged cells up-regulated numerous stress-response nuclear factors (e.g., Nrf2, FoxO3a, Ref1, TRX1) that regulate a battery of well-known adaptogens, antioxidants, mediators of autophagy and mitochondrial remodeling (e.g., HSP70, HO1, p62/SQSTM1, NFκB, Sirt3, and LC3; Fig. 3). Thus, this paper is the first report on efficiency of this network in mesenchymal stromal cells challenged with LPS. Moreover, the increase in LC3 was associated with massive formation of the LC3-positive vesicles featuring autophagosomes and autolysosomes (Fig. 4). Some of these vacuoles can be identified as secretory autolysosomes by the presence of multilamellar structures (most likely fibers of collagen) released extracellularly, while others contained fractured organelles including compromised mitochondria, which underwent degradation in large-size autolysosomes (Figs. 4 and 5).

The LPS-induced inflammatory effects in MSCs were associated with dramatic remodeling of the mitochondrial network (Figs. 5 and 6). This remodeling was characterized by (i) activation of fission/fusion events, and (ii) increase in the number of aberrant mitochondria and mitophagy events. All the above data suggest that MSCs challenged with LPS can employ a battery of complex adaptive responses including remodeling mitochondrial platform that ultimately enable them to resist to the inflammatory stress and damage to cellular constituents.

 

References:

[1]. West AP, Brodsky IE, Rahner C, Woo DK, Erdjument-Bromage H, Tempst P, Walsh MC, Choi Y, Shadel GS, Ghosh S. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature. 2011;472(7344):476-80.

[2]. Singh SB, Ornatowski W, Vergne I, Naylor J, Delgado M, Roberts E, Ponpuak M, Master S, Pilli M, White E, Komatsu M, Deretic V. Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria. Nat Cell Biol. 2010 12(12):1154-65.

[3]. Tait SW, Green DR. Mitochondria and cell signalling. J Cell Sci. 2012 125(Pt 4):807-15

[4]. Brigelius-Flohé R, Flohé L. Basic principles and emerging concepts in the redox control of transcription factors. Antioxid Redox Signal. 2011;15(8):2335-81.

[5]. Calabrese V, Cornelius C, Dinkova-Kostova AT, Iavicoli I, Di Paola R, Koverech A, Cuzzocrea S, Rizzarelli E, Calabrese EJ. Cellular stress responses, hormetic phytochemicals and vitagenes in aging and longevity. Biochim Biophys Acta. 2012; 1822(5):753-83.

Gorbunov_etal_Fig_1_120213Fig 1. Immunoblot analysis of expression of NFκB and iNOS  proteins in MSCs subjected to LPS challenge.

Panel A. Immunoblotting bands of NFκB and iNOS.

Panel B. Densitometry histograms of iNOS bands in MSCs subjected to challenge with LPS. The presented bars indicate the relative density of iNOS protein (normalized to density of actin bands). The statistical significance was determined by Student’s t-test (n=3).

Conditions: MSCs were incubated with 500 ng/ml LPS for 3 h. The cells were harvested at 24 h following challenge with LPS.

Gorbunov_etal_Fig_2_120213Fig 2. Confocal immunofluorescence imaging of the DhRho 123–detectable ROS/RNS products in MSCs challenged with LPS.

Panel A1 – Projection of oxidized form of DhRho 123 (Rho 123) (green channel) in control MSCs; Panel A2 – Overlay of projection of Rho 123 shown in A1 and a respective DIC image.

Panel B1 – Projection of Rho 123 (green channel) in LPS-challenged MSCs; Panel B2 – Overlay of projection of Rho 123 shown in B1 and a respective DIC image. A dramatic increase in Rho 123 fluorescence occurred in the LPS-challenged MSCs.

Panel C1 – Projection of Rho 123 (green channel) in LPS-challenged MSCs and treated with LNIL, an iNOS inhibitor; Panel C2 – Overlay of projection of Rho 123 shown in C1 and a respective DIC image. Suppression of Rho 123 fluorescence occurred in the LPS-challenged MSCs.

Bright green fluorescence of the ROS-activated Rho 123 shown in mitochondria is shown with red arrows. Diffused green fluorescence of the ROS/RNS-activated Rho 123 in the cytoplasm is shown with white arrows in panel B.

The confocal images were taken with pinhole setup to obtain 0.5 µm Z-sections. The experimental conditions are indicated in Fig. 1.

Gorbunov_etal_Fig_3_120213Fig 3. Western immunoblot analysis of redox-response and autophagy-mediated proteins in MSCs challenged  with LPS.

Panel A. Representative immunoblotting bands of HSP70, Nrf2, p62/SQSTM1, Sirt3, and LC3 proteins. The protein extracts were obtained from MSC cultures 24 h after challenge with LPS.

Panels B and C. Representative immunoblotting bands of Hemeoxygenase 1 (HO1) protein (B) and respective densitometry histograms of HO1 bands (C) in MSCs stimulated with LPS. The presented bars indicate the relative density of HO1 protein (normalized to density of actin bands). The statistical significance was determined by Student’s t-test (n=3). The experimental conditions are indicated in Fig. 1.

Gorbunov_etal_Fig_4_120213Fig 4. Assessment of autophagosome formation in MSCs challenged with LPS.

Panels A – D  Confocal immunofluorescence imaging

Panel A – (Green channel). LC3 projection in control MSCs.

Panel B – Overlay of projections of LC3 (green channel), iNOS (red channel), and nuclei (blue channel) in control MSCs.

Panel C – (Green channel). LC3 projection in MSCs challenged with LPS.

Panel D – Overlay of projections of LC3 (green channel), iNOS (red channel), and nuclei (blue channel) in MSCs challenged with LPS.  Spatial localization of LC3 is indicated with white arrows.

The experimental conditions are indicated in Fig. 1. Counterstaining of nuclei was with Hoechst 33342 (blue channel). The confocal images were taken with pinhole setup to obtain 0.5 µm Z-sections.

Panel E is TEM image of MSCs challenged with LPS. Autophagosome (ATG) membranes are indicated with yellow arrows; autophagy of aberrant mitochondria is indicated with white arrow; fusion of lysosomes with ATG is indicated with pink arrow; an mitochondrial spheroid is indicated with a green arrow.

Gorbunov_etal_Fig_5_120213Fig. 5. Transmission electron (TEM) analysis of mitochondrial remodeling in MSCs challenged with LPS.

Panel A:  Image of a control MSC. Mitochondria are indicated with pink arrows.

Panels B-F – Images of MSCs challenged with LPS.

Panel B – Aberrant mitochondrion remodeling is shown with a red arrow. Mitochondrial spheroids are indicated with green arrows. Mt, – mitochondrion. ATG – autophagosome.

Panels C and D – Fusion of damaged mitochondria (Mt) with autophagosomes (ATG) is indicated with red arrows. ATG membranes are indicated with white arrows. Mitochondrial spheroids are indicated with green arrows. Mitochomdrial remodeling is indicated with a yellow arrow.

Panel E – Fusion of mitochondria (Mt) is indicated with a yellow arrow.

Panel F – Formation of elongated mitochondria is indicated with a yellow arrow. Mitophagy is indicated with a red arrow.

The experimental conditions are indicated in Fig. 1. The cells were fixed for the analyses at 24 h after challenge with LPS.

Gorbunov_etal_Fig_6_120213Fig. 6. Confocal immunofluorescence imaging of mitochondrial remodeling in MSCs challenged with LPS.

Mitochondrial networks were visualized using projections of TOM20 (red channel), a mitochondrial marker.

Panels A and B – Control MSCs: mitochondrial network is presented by small-size dots.

Panels C and D – MSCs challenged with LPS: formation of long-length mitochondrial network occurred due to mitochondrial fusion.

The experimental conditions are indicated in Fig. 1.  The cells were fixed for analyzes at 24 h after challenge with LPS. Counterstaining of nuclei was with Hoechst 33342 (blue channel). The confocal images were taken with pinhole setup to obtain 0.5 µm Z-sections.

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