MOTS-c is a mitochondrial-encoded interferon-linked host defense peptide
- Leonard Davis School of Gerontology, University of Southern California, United States
- USC Earth Sciences, United States
- USC Norris Comprehensive Cancer Center, United States
- USC Stem Cell Initiative, United States
- Molecular and Computational Biology Department, USC Dornsife College of Letters, Arts and Sciences, United States
- Biochemistry and Molecular Medicine Department, USC Keck School of Medicine, United States
- Biomedical Science, Graduate School, Ajou University, Republic of Korea
Figures
Figure 1 with 4 supplements
MOTS-c is a mitochondrial-encoded host defense peptide (HDP).
(A) Bacteria and bacteria-derived mitochondria possess gene-encoded immune peptides, known as HDPs in higher eukaryotes. (B) MOTS-c has a hydrophobic core (8YIFY11), determined using the hydrophobicity scales of Kyte and Doolittle, 1982, and Sweet and Eisenberg, 1983. Blue: cationic residues. (C) MOTS-c has a cationic tail (13RKLR16) that confers positive charge (Z) across a pH range. (D) MOTS-c treatment (0–100 µM) immediately aggregates E. coli in a dose-dependent manner. (E) MOTS-c-dependent E. coli aggregation is lost in increasing salt concentrations (NaCl; 0–1%) and (F, G) requires its hydrophobic core and cationic tail, consistent with other HDPs. EGFP-expressing E. coli (BL21) is shown. Wild-type MOTS-c (WT) and mutants devoid of its hydrophobic (YIFY: 8YIFY11>8AAAA11) or cationic domain (RKLR: 13RKLR16>13AAAA16). Bar, 75 µm. (H) Scanning electron micrographs of E. coli treated with MOTS-c (100 µM) for 0 (immediate fixation), 30, and 60 min (n=3). Representative images are shown. Bar, 100 nm. (I–J) Growth curve of E. coli (BL21), measured by optical density at 600 nm (OD600), following (n=6) (I) MOTS-c treatment in the presence of 1% NaCl, and (J) treatment with wild-type (WT) MOTS-c and mutants devoid of its hydrophobic (8YIFY11>8AAAA11; YIFY), or cationic domain (13RKLR16>13AAAA16; RKLR). Data are expressed as mean ± SEM. Two-way ANOVA repeated measures. ***p<0.001.
Figure 1—figure supplement 1
The effect of MOTS-c bacterial aggregation.
(A) MOTS-c (100 μM) was added to an E. coli suspension in media (water), which immediately aggregated and precipitated bacteria. MOTS-c levels remaining in media (water) and associated with E. coli were detected by western blotting. MOTS-c (100 μM) levels in media (water) without any bacteria are also shown as reference. (B) MOTS-c immediately agglutinates bacteria in a dose-dependent manner. E. coli and methicillin-resistant Staphylococcus aureus (MRSA) were resuspended in water and treated with MOTS-c at varying doses (n=6). Bar, 200 μm. (C) E. coli and MRSA were collected at various points of growth (i.e. lag, early/mid/late log, and stationary phase), washed and resuspended in water, then treated with MOTS-c (100 μM). Images were taken immediately and rendered equally across all panels to aid in visual recognition of bacteria (n=3). Bar, 200 μm. (D) E. coli and MRSA were treated with wild-type (WT) or mutant MOTS-c (100 μM) and imaged immediately (n=6). MOTS-c mutants were devoid of its hydrophobic (8YIFY11>8AAAA11; MOTS-cYIFY) or cationic domain (13RKLR16>13AAAA16; MOTS-cRKLR). Bar, 400 μM.
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Figure 1—figure supplement 1—source data 1
PDF of uncropped western blots with relevant bands indicated.
- https://cdn.elifesciences.org/articles/87615/elife-87615-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Original western blot TIF files from ChemiDoc.
- https://cdn.elifesciences.org/articles/87615/elife-87615-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
MOTS-c exhibits specificity in bacterial targeting.
MOTS-c (100 μM) treatment causes immediate aggregation of E. coli but not Salmonella typhimurium or Pseudomonas aeruginosa (n=6). Representative image is shown. See Figure 1D.
Figure 1—figure supplement 3
MOTS-c targets methicillin-resistant S. aureus (MRSA) membranes.
Scanning electron micrographs of MRSA treated with MOTS-c (100 μM) for 0 (immediate fixation), 30, and 60 min (n=3). Representative images are shown. Bar, 100 nm.
Figure 1—figure supplement 4
The effect of MOTS-c on bacterial metabolism and growth.
(A) Membrane integrity assessment by time-course quantification of SYTOX Green nucleic acid stain, which is excluded by intact E. coli membranes (n=6). (B) Total cellular ATP levels measured following MOTS-c treatment (100 μM) in E. coli (n=3). RLU: relative light units. (C) Real-time metabolic assessment of E. coli respiration (oxygen consumption rate [OCR]) and glycolysis (extracellular acidification rate [ECAR]) (n=8). (D) Growth curve of E. coli, measured by optical density at 600 nm (OD600) following various doses of MOTS-c peptide treatment (n=6), (E) E. coli growth in M9 media with/without MOTS-c treatment (100 μM) was assessed using alamarBlue, a redox-sensitive resazurin dye (n=3). RFU: relative fluorescence units. (F) E. coli were transformed with expression vectors harboring the MOTS-c ORF (open reading frame) that can be induced by IPTG (isopropyl β-D-1-thiogalactopyranoside). Growth determined by optical density at 600 nm (OD600) periodically for 8 hr (n=3). Data expressed as mean ± SEM. Mann-Whitney test for (B, E) and two-way ANOVA (repeated measures) (A, C, D, and F). **p<0.01, ***p<0.001.
Figure 2 with 1 supplement
MOTS-c enhances survival from methicillin-resistant S. aureus (MRSA) exposure in vivo.
(A–O) 6×108 colony-forming unit (CFU) of mid-log phase MRSA either resuspended in (i) 100 µM MOTS-c, (ii) vehicle (water), or (iii) in vehicle and then heat-killed in a water bath. MRSA preparations were then immediately injected IP into 6-month-old female C57BL/6J mice (n=5–6). Mice were euthanized and blood collected after 6 and 72 hr. (A) Survival and (B) weight were monitored for 72 hr. (C) 6×108 CFU of MRSA resuspended in 100 µM MOTS-c or water was serially diluted and plated on LB agar before injection and colonies counted after overnight incubation. (D–L) Cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IFN-γ, and TNF-α) were measured in plasma by multiplex ELISA after 6 hr (n=5) and 72 hr (n=1 for control and n=6 for others; surviving mice). Plasma levels of (M) blood urea nitrogen (BUN), (N) aspartate transaminase (AST), and (O) alanine transaminase (ALT) were measured by ELISA at 6 hr (n=5) and 72 hr (n=1 for control and n=6 for others). Data are expressed as mean ± SEM. Log-rank (Mantel-Cox) test for (A), two-way ANOVA (repeated measures) for (B), Mann-Whitney test for (C), and Kruskal-Wallis test (6 hr time point) and Mann-Whitney test (72 hr time point; due to only one control surviving) for (D–O). *p<0.05, **p<0.01.
Figure 2—figure supplement 1
MOTS-c enhances survival from methicillin-resistant S. aureus (MRSA) exposure in vivo in male mice.
(A–C) 4×108 colony-forming unit (CFU) of mid-log phase MRSA was resuspended in 100 µM MOTS-c or vehicle (water) and immediately injected IP into 3.5- to 4-month-old male C57BL/6J mice (n=4–5). Survival (A) and weight (B) were monitored for 72 hr. (C) 4×108 CFU of MRSA resuspended in 100 µM MOTS-c or water was serially diluted and plated on LB agar before injection and colonies counted after overnight incubation. (D–E) 4×108 CFU of mid-log phase S. aureus (MRSA) was resuspended in phosphate-buffered saline (PBS) and either kept on ice (control) or heat-killed in a 70°C water bath for 20 min. Live or heat-killed MRSA was injected IP into 3.5- to 4-month-old male C57BL/6J mice (n=10). Survival (D) and weight (E) were monitored for 72 hr. Data expressed as mean ± SEM. Log-rank (Mantel-Cox) test (A,D), Mann-Whitney test (C), and two-way ANOVA (repeated measures) (E).
Figure 3 with 1 supplement
MOTS-c is induced in activated and differentiating monocytes.
(A–B) Total endogenous MOTS-c levels measured as a function of time following monocyte differentiation in (A) primary human monocytes by macrophage colony-stimulating factor (M-CSF) (100 ng/ml) (n=3) and (B) THP-1 cells by phorbol myristate acetate (PMA) (15 nM) (n=6). (C–E) Total endogenous MOTS-c levels following THP-1 monocyte activation by lipopolysaccharides (LPS) (100 ng/ml) and interferon gamma (IFNγ) (20 ng/ml) (C) in combination (n=6), (D) LPS alone (n=6), and (E) IFNγ alone (n=12). Data are expressed as mean ± SEM. Mann-Whitney test. *p<0.05, **p<0.01, ****p<0.0001.
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Figure 3—source data 1
PDF of uncropped western blots with relevant bands indicated.
- https://cdn.elifesciences.org/articles/87615/elife-87615-fig3-data1-v1.zip
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Figure 3—source data 2
Original western blot TIF files from ChemiDoc.
- https://cdn.elifesciences.org/articles/87615/elife-87615-fig3-data2-v1.zip
Figure 3—figure supplement 1
Endogenous MOTS-c levels in macrophages.
(A) Total endogenous MOTS-c levels in differentiated macrophages (THP-1) measured following lipopolysaccharides (LPS) treatment for 16 hr. (B) Comparison of endogenous MOTS-c levels in macrophages (THP-1; 15 μg total cell lysates/well) and measured synthetic peptide levels (1 and 5 μg). Antibodies were competed out with MOTS-c peptide to confirm detection specificity. Synthetic MOTS-c peptide was chemically synthesized without any post-translational modifications (PTM) or oligomerizations.
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Figure 3—figure supplement 1—source data 1
PDF of uncropped western blots with relevant bands indicated.
- https://cdn.elifesciences.org/articles/87615/elife-87615-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original western blot TIF files from ChemiDoc.
- https://cdn.elifesciences.org/articles/87615/elife-87615-fig3-figsupp1-data2-v1.zip
Figure 4 with 1 supplement
MOTS-c reprograms early nuclear gene expression during monocyte differentiation.
(A) A time-course measurement of endogenous MOTS-c in purified nuclear extracts following THP-1 monocyte differentiation by phorbol myristate acetate (PMA) (15 nM). (B) Confocal fluorescence images of THP-1 monocytes treated with FITC-MOTS-c (1 µM) for 30 min, showing nuclear localization. Nucleus marked by DAPI staining. Bar, 10 µm. (C–H) THP-1 monocytes were primed with MOTS-c (10 µM) or vehicle control for 2 hr, then differentiated with PMA±MOTS-c (10 µM) for 2 hr, at which time RNA was collected for bulk RNA-seq analysis (n=6); false discovery rate (FDR)<5%. (C) Multidimensional scaling (MDS) analysis across control, PMA, and PMA+MOTS-c groups based on RNA-seq expression profiles after DESeq2 VST normalization. (D) Heatmap of significantly differentially regulated genes by MOTS-c by DESeq2 analysis. (E) Protein-protein interaction network analysis based on genes that were significantly differentially up- and downregulated by MOTS-c (FDR<5%) using the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database version 11.0 (Szklarczyk et al., 2019). (F) Significantly enriched biological functions based on gene set enrichment analysis (GSEA) using Gene Ontology (GO). Selected groups are shown, full data in Supplementary file 1. (G) Correlation plot of gene expression changes by DESeq2 upon (i) regular induction of differentiation (control vs. PMA) compared to (ii) MOTS-c-directed induction of differentiation (PMA vs. PMA+MOTS-c). Spearman rank correlation (Rho) and significance of this correlation are reported. Genes that are significantly regulated only upon MOTS-c treatment but not during normal differentiation are highlighted in red and may underlie a specific MOTS-c-induced macrophage state. (H) Protein-protein interaction network analysis based on the 64 genes that were significantly differentially up- or downregulated by MOTS-c (FDR<5%) as described in (G) using the STRING database version 11.0 (Szklarczyk et al., 2019). MΦ=macrophage.
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Figure 4—source data 1
PDF of uncropped western blots with relevant bands indicated.
- https://cdn.elifesciences.org/articles/87615/elife-87615-fig4-data1-v1.zip
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Figure 4—source data 2
Original western blot TIF files from ChemiDoc.
- https://cdn.elifesciences.org/articles/87615/elife-87615-fig4-data2-v1.zip
Figure 4—figure supplement 1
Nuclear MOTS-c in monocytes during activation and exogenous MOTS-c treatment.
(A) MOTS-c translocates to the nucleus upon activation by lipopolysaccharides (LPS)+interferon gamma (IFNγ). A time-course measurement of endogenous MOTS-c in purified nuclear extracts following monocyte (THP-1) activation by LPS+IFNγ. Lamin B1 was used as a loading control for purified nuclear samples. See also Figure 4A. (B) Time-course detection of intracellular MOTS-c-FLAG peptide (10 µM) following treatment in THP-1 monocytes. (C) Intracellular levels of MOTS-c-FLAG peptide (10 µM) following treatment in THP-1 monocytes at different temperatures (i.e. 4°C vs. 37°C) for 30 min. (D) Exogenously treated MOTS-c peptide enters monocytes and localizes to the nucleus. Confocal fluorescence images of monocytes (THP-1) treated with FITC-MOTS-c (1 μM) for 30 min, showing nuclear localization. Nucleus marked by DAPI staining. Bar, 10 μm.
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Figure 4—figure supplement 1—source data 1
PDF of uncropped western blots with relevant bands indicated.
- https://cdn.elifesciences.org/articles/87615/elife-87615-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Original western blot TIF files from ChemiDoc.
- https://cdn.elifesciences.org/articles/87615/elife-87615-fig4-figsupp1-data2-v1.zip
Figure 5 with 1 supplement
MOTS-c promotes the generation of macrophages with enhanced antibacterial capacity.
(A) Primary human monocytes were differentiated by macrophage colony-stimulating factor (M-CSF) for 3 days with MOTS-c (10 µM) or vehicle (ddH2O) (2 hr priming, then single treatment with M-CSF). Representative images of adhered macrophages (n=6; MΦ=macrophage; bar, 10 µm). (B–F) THP-1 macrophages were differentiated for 4 days with/without MOTS-c treatment (10 µM; 2 hr priming, then single treatment with phorbol myristate acetate [PMA]). (B) Gentamicin protection assay in MOTS-c-programmed THP-1 macrophages following 1.5 or 3 hr post-infection of E. coli (MOI: 10). CFU: colony-forming units (n=6). (C–F) MOTS-c-programmed THP-1 macrophages were stimulated with lipopolysaccharides (LPS) (100 ng/ml) and (C) secreted levels of IL-1β, IL-1Ra, and TNF-α measured by ELISA after 20 hr (n=6), (D) cytokine expression levels determined by RT-qPCR after 16 hr (n=6), and (E–F) metabolic flux assessed (n=15) by cellular respiration (oxygen consumption rate [OCR]) (E) immediately after LPS stimulation and (F) 16 hr after LPS stimulation. Data are expressed as mean ± SEM. Mann-Whitney test, except for (E, F), which used two-way ANOVA repeated measures. *p<0.05, **p<0.01, ***p<0.001.
Figure 5—figure supplement 1
The presence of MOTS-c during monocyte-derived macrophage differentiation generates macrophages with an altered phenotype.
(A) MOTS-c affects primary human monocyte differentiation. Primary human monocytes were differentiated with macrophage colony-stimulating factor (M-CSF) for 6 days with MOTS-c (10 μM) treatment or vehicle (ddH2O) only during the first 24 hr (n=6; MΦ=macrophage; bar, 10 μm). Adherent macrophages were imaged and counted. See also Figure 5A. (B) Macrophages (THP-1) were differentiated with MOTS-c (10 μM) or vehicle (ddH2O) (n=6) for 4 days. Time-course gene expression levels in MOTS-c-programmed macrophages by qPCR of cytokines CCL2, CCL3, CCL5, CXCL9, CXCL10, CXCL11, IL6, IL10, IL12A, and IL12B following lipopolysaccharides (LPS) treatment (100 ng/ml) (0, 6, and 16 hr). Relative gene expression, calculated based on the 0 time point, is shown. RQ: relative quantification. See also Figure 5D. Data expressed as mean ± SEM. Mann-Whitney test, *p<0.05, **p<0.01.
Figure 6 with 4 supplements
MOTS-c generates unique macrophages characterized by enhanced interferon signaling and antigen presentation in an age-related manner.
(A) Single-cell RNA-seq (scRNA-seq) was performed on bone marrow-derived macrophages (BMDMs) from young (4 mo.) and old (20 mo.) mice of both sexes that were differentiated for 7 days in the presence of MOTS-c (10 µM) or vehicle (ddH2O), treated once concomitantly with first exposure to M-CSF, and present only for the first 3 days of differentiation. (B) Multidimensional scaling (MDS) analysis across each of the eight groups based on pseudobulk gene expression profiles for each biological group after performing DESeq2 VST normalization. (C–D) Uniform manifold approximation and projection (UMAP) plot on (C) all mice and (D) separated by age and sex, with cells color-coded based on shared nearest neighbor (SNN) clustering. Clusters 5 and 6 were enriched in MOTS-c-programmed BMDM populations. (E) Box plot of relative cluster cell proportion ratios between MOTS-c-programmed vs. control macrophages across clusters. Note that clusters 5 and 6 are consistently found in higher proportion in MOTS-c-treated samples compared to their corresponding control condition. (F) Dotplot of select genes enriched in clusters 5 and 6 (see also Figure 6—figure supplements 2–4 and Supplementary file 3). (G) Heatmap of the top 10 differential gene markers of each of the eight clusters in BMDMs induced in the presence/absence of MOTS-c (false discovery rate [FDR]<5%).
Figure 6—figure supplement 1
Pairwise distance analysis between female and male samples in control and MOTS-c-treated conditions for bone marrow-derived macrophage (BMDM) pseudobulk transcriptomes.
The distance between each pair of (female, male) pseudobulk sample is computed as 1–Rho for each pair (YF-YM, YF-OM, OF-YM, and OF-OM) in the control and MOTS-c-treated conditions. To test whether the distance between (female, male) pseudobulk sample pair is reduced upon MOTS-c treatment, we used a one-sided paired Wilcoxon rank-sum test (p~0.0625). YF = young female; YM = young male; OF = old female; OM = old male.
Figure 6—figure supplement 2
Genes enriched in MOTS-c-programmed macrophage populations.
Single-cell RNA-seq (scRNA-seq) was performed on bone marrow-derived macrophages (BMDMs) from young (4 mo.) and old (20 mo.) mice of both sexes that were differentiated for 7 days with MOTS-c (10 μM) or vehicle (ddH2O). MOTS-c was given only once concomitantly with macrophage colony-stimulating factor (M-CSF) at the onset of differentiation, and the media was replaced after 3 days in both the control- and MOTS-c-treated conditions (see also Figure 6A). Volcano plots on differentially expressed genes in clusters 5 and 6 that are enriched by MOTS-c are shown here (see also Figure 6D–F, Figure 6—figure supplements 3 and 4, and Supplementary file 3).
Figure 6—figure supplement 3
MOTS-c generates macrophages that uniquely express genes related to antigen presentation and interferon signaling.
Single-cell RNA-seq (scRNA-seq) was performed on bone marrow-derived macrophages (BMDMs) from young (4 mo.) and old (20 mo.) mice of both sexes that were differentiated for 7 days with MOTS-c (10 μM) or vehicle (ddH2O). MOTS-c was given only once concomitantly with macrophage colony-stimulating factor (M-CSF) at the onset of differentiation, and the media was replaced after 3 days in both the control- and MOTS-c-treated conditions (see also Figure 6A). Dotplot of genes enriched in each of the eight clusters, showing MOTS-c-programmed BMDMs (clusters 5 and 6) have a unique gene expression profile not present in other clusters (see also Figure 6D–F, Figure 6—figure supplements 2 and 4, and Supplementary file 3).
Figure 6—figure supplement 4
MOTS-c-programmed macrophage populations exhibit differential regulation of antigen presentation and interferon-related pathways.
Single-cell RNA-seq (scRNA-seq) was performed on bone marrow-derived macrophages (BMDMs) from young (4 mo.) and old (20 mo.) mice of both sexes that were differentiated for 7 days with MOTS-c (10 μM) or vehicle (ddH2O). MOTS-c was given only once concomitantly with macrophage colony-stimulating factor (M-CSF) at the onset of differentiation, and the media was replaced after 3 days in both the control- and MOTS-c-treated conditions (see also Figure 6A). Dotplots of biological processes derived from scRNA-seq data for clusters 5 and 6, based on gene set enrichment analysis (GSEA) of Gene Ontology Biological Process (GO_BP) at false discovery rate (FDR)<5% (see also Figure 6D–F, Figure 6—figure supplements 2 and 3, and Supplementary file 3).
Author response image 1
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Videos
Video 1
Aggregation of E. coli by MOTS-c.
A 1 ml aliquot of E. coli BL21 culture (OD600 ≈ 0.6) was pelleted by centrifugation at 6000×g and resuspended in ddH2O. MOTS-c was immediately added to a final concentration of 100 μM, and the suspension was vortex-mixed and transferred to a cuvette containing 1 ml of ddH2O for imaging.
Additional files
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Supplementary file 1
Differentially regulated genes by phorbol myristate acetate (PMA) and MOTS-c in THP-1 cells.
- https://cdn.elifesciences.org/articles/87615/elife-87615-supp1-v1.xlsx
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Supplementary file 2
Gene set enrichment analysis (GSEA) of genes differentially regulated by MOTS-c in THP-1 cells.
- https://cdn.elifesciences.org/articles/87615/elife-87615-supp2-v1.xlsx
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Supplementary file 3
Genes enriched in clusters 5 and 6 in single-cell RNA-seq (scRNA-seq) of young and old bone marrow-derived macrophages of both sexes.
- https://cdn.elifesciences.org/articles/87615/elife-87615-supp3-v1.xlsx
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Supplementary file 4
Gene set enrichment analysis (GSEA) of genes enriched in clusters 5 and 6 in single-cell RNA-seq (scRNA-seq) of young and old bone marrow-derived macrophages of both sexes.
- https://cdn.elifesciences.org/articles/87615/elife-87615-supp4-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/87615/elife-87615-mdarchecklist1-v1.docx
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MOTS-c is a mitochondrial-encoded interferon-linked host defense peptide
eLife 12:RP87615.
https://doi.org/10.7554/eLife.87615.3
