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. 2023 Feb:59:102571.
doi: 10.1016/j.redox.2022.102571. Epub 2022 Dec 8.

Loss of selenoprotein W in murine macrophages alters the hierarchy of selenoprotein expression, redox tone, and mitochondrial functions during inflammation

Sougat Misra  1 , Tai-Jung Lee  1 , Aswathy Sebastian  2 , John McGuigan  1 , Chang Liao  3 , Imhoi Koo  4 , Andrew D Patterson  5 , Randall M Rossi  6 , Molly A Hall  1 , Istvan Albert  2 , K Sandeep Prabhu  7
Affiliations

Loss of selenoprotein W in murine macrophages alters the hierarchy of selenoprotein expression, redox tone, and mitochondrial functions during inflammation

Sougat Misra et al. Redox Biol. 2023 Feb.

Abstract

Macrophages play a pivotal role in mediating inflammation and subsequent resolution of inflammation. The availability of selenium as a micronutrient and the subsequent biosynthesis of selenoproteins, containing the 21st amino acid selenocysteine (Sec), are important for the physiological functions of macrophages. Selenoproteins regulate the redox tone in macrophages during inflammation, the early onset of which involves oxidative burst of reactive oxygen and nitrogen species. SELENOW is a highly expressed selenoprotein in bone marrow-derived macrophages (BMDMs). Beyond its described general role as a thiol and peroxide reductase and as an interacting partner for 14-3-3 proteins, its cellular functions, particularly in macrophages, remain largely unknown. In this study, we utilized Selenow knock-out (KO) murine bone marrow-derived macrophages (BMDMs) to address the role of SELENOW in inflammation following stimulation with bacterial endotoxin lipopolysaccharide (LPS). RNAseq-based temporal analyses of expression of selenoproteins and the Sec incorporation machinery genes suggested no major differences in the selenium utilization pathway in the Selenow KO BMDMs compared to their wild-type counterparts. However, selective enrichment of oxidative stress-related selenoproteins and increased ROS in Selenow-/- BMDMs indicated anomalies in redox homeostasis associated with hierarchical expression of selenoproteins. Selenow-/- BMDMs also exhibited reduced expression of arginase-1, a key enzyme associated with anti-inflammatory (M2) phenotype necessary to resolve inflammation, along with a significant decrease in efferocytosis of neutrophils that triggers pathways of resolution. Parallel targeted metabolomics analysis also confirmed an impairment in arginine metabolism in Selenow-/- BMDMs. Furthermore, Selenow-/- BMDMs lacked the ability to enhance characteristic glycolytic metabolism during inflammation. Instead, these macrophages atypically relied on oxidative phosphorylation for energy production when glucose was used as an energy source. These findings suggest that SELENOW expression in macrophages may have important implications on cellular redox processes and bioenergetics during inflammation and its resolution.

Keywords: Arginase; Energy metabolism; Glycolysis; Krebs cycle; Metabolism; Mitochondrial respiration; Reactive oxygen species; Resolution; Sik2.

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Conflict of interest statement

Conflict-of-interest statement All the authors have read the manuscript and approved its final version before the submission. None of the authors have any potential conflict-of-interests to declare.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
A schematic illustration depicting the experimental design to elucidate the differences in inflammatory response in WT and Selenow−/− BMDMs. The immunoblots confirms complete loss of SELENOW in Selenow−/− BMDMs.
Fig. 2
Fig. 2
A. Expression (RNAseq, normalized count data) of 18 selenoprotein genes in WT and Selenow−/− BMDMs following treatment with 100 ng/ml LPS (n = 3 for each condition, except for KO 24 h for which only 2 replicates were available for analyses). Using the Dseq2 pipeline, the normalized expression data from RNA-seq experiment were extracted and plotted. Statistical significance was computed using a linear model, as described in the Materials and Methods section. Data have been presented as mean ± standard error of mean (SEM). GE indicates ‘genotype effect’ from two-way ANOVA analysis and the values indicates corresponding statistical significance. Mean values with asterisks (*) indicates statistically significant difference between WT and Selenow−/− BMDMs at a given time point. Alpha (α) and beta (β) indicate statistically significant difference between time point 0 h and other time points of a measured variable in WT and Selenow−/−, respectively. Denoted p values are as follows: */α/β = ≤0.05; **/αα/ββ = ≤0.01; ***/ααα/βββ = ≤0.001. Identical notations of statistical significance have been used throughout the manuscript. B. Representative immunoblots of selected selenoproteins in WT and Selenow−/− BMDMs following treatment with 100 ng/ml LPS (n = 3). C. Semi-quantitative analyses of selenoprotein expression as outlined above. Band intensity was measured using Image J software (n = 3).
Fig. 3
Fig. 3
A. A schematic diagram describing the pathway involved in the biogenesis of selenoproteins mRNAs and subsequent translation of mRNAs into nascent polypeptides of selenoproteins. B. Expression (RNAseq, normalized count data) of 15 genes that are associated with the biosynthesis of selenoproteins in WT and Selenow−/− BMDMs following treatment with 100 ng/ml LPS. Although, the involvement of Sephs1 in selenoproteins biosynthesis is not well-characterized, we also probed its expression during sterile inflammation. For the details on the statistical analyses, please refer to Fig. 2A legend. C.Top panel, Immunoblot of SEPHS2 from whole-cell lysate of WT and Selenow−/− BMDMs following treatment with 100 ng/ml LPS (n = 3). Bottom panel, Semi-quantitative analyses of SEPHS2 expression.
Fig. 4
Fig. 4
A. Flow cytometry analysis of the frequency of ROS-positive cells in WT and Selenow−/− BMDMs following treatment with LPS (100 ng/ml). Data from 3 independent experiments were concatenated and presented as is. Histograms demonstrate the relative frequency of ROS-positive viable cells (n = 3). B. Median CellRox ROS intensity of WT and Selenow−/− BMDMs (top panel) and the area under the curve (bottom panel) of ROS intensity from the same experiments, as outlined above (n = 3). SEM values were not calculated for CellRox ROS intensity data as samples were concatenated for analyses. Statistical significance of AUC for ROS intensity was computed using unpaired t-test.
Fig. 5
Fig. 5
A. Temporal changes in the expression of selected genes of inflammatory pathway that were differentially expressed between WT and Selenow−/− BMDMs following treatment with 100 ng/ml LPS. Detailed multiple comparisons between the groups from two-way ANOVA are summarized in the Supplementary Table ST1. For the details on the statistical analyses, please refer to Fig. 2A legend. B and C. Representative immunoblots and semi-quantitative analyses of FAH, SIK1, SIK2, and ARG1 expression in BMDMs lysates (n = 3). D. Schematic representation of pathway involved in arginine metabolism. E. Normalized abundance of arginine, ornithine, citrulline, fumarate, and aspartate in WT and Selenow−/− BMDMs following treatment with LPS (n = 3). F. Ornithine to arginine and citrulline to arginine ratio in WT and Selenow−/− BMDMs. For the details on the statistical analyses, please refer to Fig. 2A legend (n = 3). G. Left panel, Efferocytosis in WT and Selenow−/− BMDMs macrophages that were co-cultured with PKH26-stained apoptotic/dying neutrophils for 4 h without any stimulation. Statistical significance was computed using unpaired t-test (n = 3). Right panel, Representative flow sight image demonstrating a macrophage that engulfed a PKH26-labeled neutrophil via efferocytosis.
Fig. 6
Fig. 6
A. A real-time recording of oxygen consumption rates (OCR) in WT and Selenow−/− BMDMs following treatment with 100 ng/ml LPS (n = 5) for 1 h followed by sampling at the indicated time points (n = 5). Abbreviation, FCCP – Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, Rot – Rotenone, AA - antimycin A. B. Metabolic phenotypes of BMDMs as computed from data obtained from the mitochondrial stress test, as described in Fig. 6A. C. The calculated values for basal respiration, maximal respiration, non-mitochondrial respiration, proton lean, ATP production, percent spare respiratory capacity, and coupling efficiency from the mitochondrial stress test (n = 5). For the details on the statistical analyses, please refer to Fig. 2A legend.
Fig. 7
Fig. 7
A. Qualitative assessment of the relative utilization of mitochondrial (oxidative phosphorylation) versus glycolytic pathways for energy production. OCR and extracellular acidification rates (ECAR) data are obtained from the glycolytic stress test, as outlined in Fig. 6A. B. Extracellular acidification rates in WT and Selenow−/− BMDMs following treatment with 100 ng/ml LPS (n = 3) for 1 h followed by sampling at the indicated time points (n = 3). C. The rate of glycolysis (top left), non-glycolytic acidification (top right), glycolytic capacity (bottom left), and glycolytic reserve (bottom right) measured from the outlined glycolytic stress test (n = 3). For the details on the statistical analyses, please refer to Fig. 2A legend.
Fig. 8
Fig. 8
A. A schematic demonstrating the metabolic flux of isotope-enriched carbon from U–13C-glucose involving glycolysis and TCA cycle. Isotopologue signatures from single cycle turnover of glucose into lactate via glycolysis or into oxaloacetate via TCA cycle are shown next to the corresponding metabolite. B. Distribution of unlabeled and U–13C-labeled glucose at different time points. No significant differences in the labeling indices were observed at any time point or between the genotypes (n = 3). C. The levels of pyruvate and lactate (sum of all isotopologue) in WT and Selenow−/− BMDMs following treatment with LPS for the indicated time points (n = 3). D. The normalized abundance of selected TCA cycle intermediates and aspartate in WT and Selenow−/− BMDMs following treatment with LPS. Sum of all isotopologue were used to calculate the normalized abundance (n = 3). For the details on the statistical analyses, please refer to Fig. 2A legend. E. The levels of GSH and GSH to GSSG ratio in WT and Selenow−/− BMDMs following treatment with LPS (n = 3).
Fig. 9
Fig. 9
A. Immunoblot analysis of SELENOW in lysates prepared from the isolated mitochondrial fraction of whole liver of WT mice. VDAC, GAPDH, and histone 3 served as control for mitochondrial, cytosolic, and nuclear localization, respectively. B. Immunofluorescence detection of SELENOW in WT BMDMs. Merged signal in orange pseudo-color depicts colocalization of SELENOW along with COX4 in the mitochondria, as illustrated in the inset with white arrow. Scale bar = 6 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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