MOTS-c is a mitochondrial-encoded host defense peptide (HDP).

(A) Bacteria and bacteria-derived mitochondria possess gene-encoded immune peptides, known as host defense peptides (HDPs) in higher eukaryotes. (B) MOTS-c has a hydrophobic core (8YIFY11), determined using the hydrophobicity scales of Kyte and Doolittle (K&D)32 and Sweet and Eisenberg (S&E)33. 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) shown. Wildtype 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 minutes (n=3). Representative images 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 wildtype (WT) MOTS-c and mutants devoid of its hydrophobic (8YIFY11>8AAAA11; YIFY), or cationic domain (13RKLR16>13AAAA16; RKLR). Data expressed as mean +/- SEM. Two-way ANOVA repeated measures. *** P<0.001.

MOTS-c enhances survival from MRSA exposure in vivo.

(A-O) 6×108 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 hours. (A) Survival and (B) weight were monitored for 72 hours. (C) 6×108 CFU of MRSA resuspended in 100 µM MOTS-c or water was serial 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 hours (n=5) and 72 hours (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 hours (n=5) and 72 hours (n=1 for control and n=6 for others). Data expressed as mean +/- SEM. Log-rank (Mantel-Cox) test for (A), 2-way ANOVA (repeated measures) for (B), Mann-Whitney test for (C), and Kruskal-Wallis test (6 hour timepoint) and Mann-Whitney test (72 hour timepoint; due to only 1 control surviving.) for (D-O).* P<0.05, ** P<0.01.

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 M-CSF (100 ng/ml) (n=3) and (B) THP-1 cells by PMA (15 nM) (n=6). (C-E) Total endogenous MOTS-c levels following THP-1 monocyte activation by LPS (100 ng/ml) and IFNγ (20ng/ml) (C) in combination (n=6), (D) LPS alone (n=6), and (E) IFNγ alone (n=12). Data expressed as mean +/- SEM. Mann-Whitney test. *P<0.05, ** P<0.01, **** P<0.0001.

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 PMA (15 nM). (B) Confocal fluorescence images of THP-1 monocytes treated with FITC-MOTS-c (1 µM) for 30 minutes, 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 hours, then differentiated with PMA +/- MOTS-c (10 µM) for 2 hours, 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 down-regulated by MOTS-c (FDR < 5%) using the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database version 11.090. (F) Significantly enriched biological functions based on gene set enrichment analysis (GSEA) using gene ontology (GO). Selected groups are shown, full data in Table S1. (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- and down-regulated by MOTS-c (FDR < 5%) as described in (G) using the STRING database version 11.0 90. MΦ=macrophage.

MOTS-c promotes the generation of macrophages with enhanced antibacterial capacity.

(A) Primary human monocytes were differentiated by M-CSF for 3 days with MOTS-c (10 µM) or vehicle (ddH2O)(2-hour 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-hour priming, then single treatment with PMA). (B) Gentamicin protection assay in MOTS-c-programmed THP-1 macrophages following 1.5- or 3-hours post-infection of E. coli (MOI: 10). CFU: colony forming units (n=6). (C-F) MOTS-c-programmed THP-1 macrophages were stimulated with LPS (100 ng/ml) and (C) secreted levels of IL-1β, IL-1Ra, and TNFα measured by ELISA after 20 hours (n=6), (D) cytokine expression levels determined by RT-qPCR after 16 hours (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 hours after LPS stimulation. Data 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.

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 8 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 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 Figures S10-S12 and Table S3). (G) Heatmap of the top 10 differential gene markers of each of the 8 clusters in BMDMs induced in the presence/absence of MOTS-c (FDR < 5%).