Taurine reprograms S. uberis-induced metabolic changes in mammary glands
To determine whether taurine regulates metabolism in S. uberis induced mastitis, we infected the teats of taurine-pretreated mice with S. uberis for 24 h and then collected mouse mammary glands for metabolomic analysis. As shown in Fig. S1, intragastric administration of taurine alone could partly change mammary gland metabolism in mice. The metabolites increased by taurine administration focused on amino acids (such as proline, isoleucine, tyrosine, L-cysteine, L-allothreonine), nucleic acids (xanthine, uridine monophosphate), and taurine-associated metabolite sulfuric acid. These metabolites mainly concentrated on aminoacyl-tRNA biosynthesis, sulfur metabolism, purine metabolism, valine, leucine and isoleucine biosynthesis, and cysteine and methionine metabolism (Fig. S1). Further, taurine administration significantly changed the metabolites in S. uberis-infected mammary glands (Fig. S2). S. uberis-infected mammary glands exhibited 52 different metabolites, including 7 upregulated carbohydrate-related metabolites (galactose, mannose, galactinol, N-acetyl-D-galactosamine, 6-phosphogluconate, glucoheptonate, ribose, and 5'-methylthioadenosine) and 7 amino acids or amino-acid derivatives (valine, proline, serine, leucine, L-allothreonine, creatine, and 3, 5-dihydroxyphenylglycine). 1 TCA-cycle intermediate (fumarate), 3 amino-acid metabolites (cysteine, ornithine and ascorbate) and 3 fatty acid-metabolism precursors (glycerate, 2-monpalmitin and palmitic acid) are down-regulated (Fig. 1A). These metabolites were primarily involved in the pentose phosphate pathway; pantothenate and CoA biosynthesis; arginine and proline metabolism; glycine, serine, and threonine metabolism; valine, leucine, and isoleucine biosynthesis; glutathione metabolism and aminoacyl-tRNA biosynthesis (Fig. 1B). Taurine pretreatment decreased glycerol, stearic acid, capric acid and 2-hydroxybutanoate levels, elevated several lipid metabolites (palmitic acid, lignoceric acid, and β-glycerophosphoric acid) (Fig. 1C), and influenced unsaturated fatty-acid and fatty-acid biosynthesis, fatty-acid elongation, and purine and pyrimidine metabolism (Fig. 1D). Integrated metabolite-map and pathway analysis showed lower metabolite levels of the TCA cycle (fumarate) and glutathione metabolism (cysteine, ornithine and ascorbate), along with higher metabolite levels in the pentose phosphate pathway and higher amino acid levels following S. uberis infection. These data denoted consumption of more energy and production of anti-infectious metabolites (such as glutathione, an antioxidant) in mammary tissue to resist S. uberis challenge (Fig. 1E). Taurine pretreatment mainly reduced fatty acid metabolism (2-hydroxybutanoate), promoted unsaturated fatty acid and fatty acid biosynthesis, and nucleic acid metabolism (Fig. 1E). To confirm whether these metabolic changes were reflected at a transcriptional level, we measured the expression of key genes involved in glycolysis (HK, PFK1 and GAPDH), TCA cycle (PDH, SDH), fatty acid metabolism (PPAR-γ, LIPA) and pentose phosphate pathway (G6PDH). In line with our findings, expression of these genes in mammary glands was elevated by S. uberis challenge. Taurine dropped off most of the gene expression in S. uberis infection but increased gene expression of G6PDH (Fig. 1F). These data suggest that taurine reprograms metabolism in S. uberis-infected mammary glands.
Taurine attenuates metabolic disturbances in S. uberis challenged MECs
MECs are the main functional cells in mammary glands and play important roles in the mammary gland defense system. Pretreatment with taurine alone changed taurine-associated metabolites in mouse mammary epithelial cell line (EpH4-Ev cells). β-alanine, a competitive inhibitor of taurine transporter (TauT), was decreased by taurine pretreatment. Differently, pretreatment with taurine increased intracellular taurine and taurine-associated metabolites (glutathione, sulfuric acid, etc.). These metabolites belonged to several taurine metabolism related pathways, including sulfur metabolism, taurine and hypotaurine metabolism, β-alanine metabolism, glutathione metabolism, and primary bile acid biosynthesis (Fig. S3).
To investigate whether taurine regulates MEC metabolism during S. uberis infection, EpH4-Ev cells were incubated with S. uberis for varying times and the intracellular metabolite profile was examined. Metabolites in infected and control cells formed separate clusters on PCA and OPLA-DA plots (Fig. S4A-F) and had significant changes on volcano plots over time (Fig. S4G-H), especially at 3 h post-infection (Fig. S4I). Thus, challenge with S. uberis altered the cellular metabolic profile. Taurine pretreatment altered the metabolic profile of S. uberis-infected cells over these time points (Fig. S4J-R).
Metabolic changes induced by S. uberis infection at 1 h was slight and only several metabolites changed, but an increase of glucose-6-phosphate was present (Fig. S5A-E). In contrast, 2 or 3 h of infection with S. uberis upregulated 3 glycolysis-related metabolites (glucose, pyruvate and lactate), 4 lipid-related metabolites (octadecanol, 1-hexadecanol, 1-monopalmitin, and palmitic acid), 10 amino acids (alanine, valine, glycine, threonine, isoleucine, proline, serine, beta-alanine, taurine, and ornithine), and several other carbohydrate-related metabolites (fructose, tagatose, and inositol), while the levels of citrate and α-ketoglutarate were downregulated (Fig. 2A and Fig. S6A). The alterations at 2 or 3 h post-infection included the following metabolic pathways: aminoacyl-tRNA biosynthesis; TCA cycle; pyruvate metabolism; glycolysis or gluconeogenesis; valine; leucine, and isoleucine biosynthesis; pantothenate and CoA biosynthesis; glycine, serine, and threonine metabolism; arginine and proline metabolism; alanine, aspartate, and glutamate metabolism; and glutathione metabolism (Fig. 2B and Fig. S6B).
Pretreatment with taurine at 1 h post-infection exerted a slight change in amino acid metabolism (Fig. S5C-E). In S. uberis-infected cells (2 or 3 h), taurine pretreatment decreased carbohydrate-related metabolite levels (glucose, pyruvate, lactate, citrate, α-ketoglutarate etc.) and amino acid levels (aspartate, alanine, isoleucine, β-alanine, valine, glutamate, proline), and attenuated their matched pathways (i.e., valine, leucine, and isoleucine biosynthesis; β-alanine, alanine, aspartate, and glutamate metabolism; and TCA cycle (Fig. 2C-E and Fig. S6C-E). These data suggest that taurine reprograms S. uberis-induced metabolic changes in MECs infected by S. uberis in a time dependent manner and the alterations are not totally matched with those in the mammary glands.
MECs adopt distinct metabolism in response to various microbial stimuli compared to macrophages
There are several cell types in mammary tissue including macrophages, polymorphonuclear neutrophilic leukocytes (PMN) and regulatory T cells (Treg). Metabolic reprogramming in most proinflammatory phenotype innate immune cells (i.e., dendritic cells, M1 macrophages, and NK cells) are characterized by elevated levels of TCA intermediates (succinate, citrate) and itaconate (An inflammation limited factor in proinflammatory phenotype cells inhibiting succinate dehydrogenase and causing increases of succinate and citrate (29–31). Anti-inflammatory phenotype cells (i.e., M2 macrophages, NKT cells and Treg cells) are associated with increased OXPHOS and decreased succinate and citrate (32–35). MECs involve in the occurrence and development of S. uberis-induced inflammation in our previous study (13, 15). EpH4-Ev cells incubated with S. uberis for 3 h have decreased citrate and α-ketoglutarate but increased itaconate levels (Fig. 2A and Fig. S6A). We postulated that different metabolic patterns were present in MECs and proinflammatory phenotype innate immune cells challenged by S. uberis. Inactivated S. uberis, Escherichia coli (E. coli) and lipopolysaccharide (LPS) were used to stimulate EpH4-Ev cells and RAW 264.7 macrophages. LPS-stimulated RAW 264.7 macrophages developed an increase in extracellular acidification rate (ECAR; reflects glycolysis rate) and a decrease in oxygen consumption rate (OCR; reflects mitochondrial function and OXPHOS level) levels. MECs had an increase in ECAR and OCR levels after LPS challenge (Fig. 3A-D). Both ECAR and OCR levels increased in the above 2 cell lines with inactivated E. coli and S. uberis stimulation (Fig. 3A-D). These data indicate that MECs adopt distinct metabolism in response to various microbial stimuli compared to macrophages.
Metabolic reprogramming by taurine in S. uberis infection coordinates with the energy supply and production of anabolic intermediates
Cell metabolism generates energy and intermediates used to synthesize materials required to combat pathogen infection (36). Excessive or disordered mobilization results in cellular dysfunction. We evaluated whether taurine regulated metabolism associated with energy supply and anabolic intermediates production during S. uberis infection. Taurine pretreatment lowered ECAR and OCR levels during S. uberis challenge indicating that glycolysis and OXPHOS were inhibited in S. uberis-infected MECs (Fig. 4A-B). Cells obtain energy and anabolic intermediates via biochemical reactive catalytic enzymes. Homeostasis is critical to cellular function and stability. We found that the activities of hexokinase (HK; the first and rate-limiting enzyme of glycolysis), phosphofructokinase (PFK; the rate-limiting enzyme), and lactate dehydrogenase (LDH) increased in EpH4-Ev cells challenged with S. uberis (Fig. 4C-E). The activities of pyruvate dehydrogenase (PDH), succinate dehydrogenase (SDH), and mitochondrial complex IV (key enzymes or components in OXPHOS) also elevated (Fig. 4F-H). Taurine pretreatment significantly decreased these levels in infected EpH4-Ev cells (Fig. 4C-H). These results agreed with real-time measurements of ECAR and OCR. Thus, MECs exhibit enhanced glycolysis and OXPHOS in response to S. uberis infection which are attenuated by taurine pretreatment.
To investigate whether S. uberis challenge promoted the conversion between amino acid and energy-related metabolites (such as glutamate and α-ketoglutarate) associated with a decrease in TCA intermediates, we assayed the activities of several key enzymes involved in energy and amino acid metabolism. S. uberis infection significantly increased glutamate dehydrogenase (GDH) (Fig. 4I), glutamic oxalacetic transaminase (GOT) (Fig. 4J), and glutamic-pyruvic transaminase (GPT) activity (Fig. 4K). Taurine pretreatment attenuated these changes. These results indicate that taurine reprograms S. uberis-induced metabolic changes to coordinate the energy supply and anabolic intermediate production.
Taurine balance metabolism to alleviate inflammation induced by S. uberis
S. uberis promotes pro-inflammatory mediator production in mammary glands and MECs (12, 15). EpH4-Ev cells were treated with 2-deoxy-D-glucose (2-DG; which inhibits glycolysis) and CPI-613 (which inhibits pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, thereby blocking OXPHOS) to explore the relationship between energy metabolism and the inflammatory response. Key enzymes of glycolysis and OXPHOS detection showed that these 2 carbohydrate pathways were significantly restricted in EpH4-Ev cells with 2-DG (Fig. S7A-C) and CPI-613 (Fig. S7D-F), respectively. The effects of taurine pretreatment on glycolysis and OXPHOS were similar to those observed with 2-DG or CIP-613 treatment.
Taurine pretreatment significantly decreased TNF-α and IL-1β levels in EpH4-Ev cells infected with S. uberis (Fig. 5A-B), which was comparable to that of 2-DG treatment. Similarly, pretreatment of S. uberis infected EpH4-Ev cells with both 2-DG and taurine downregulated intracellular ROS levels more than pretreatment with taurine alone (Fig. 5C). The supernatants of EpH4-Ev cells pretreated with taurine or glycolysis inhibitors and later infected with S. uberis had lower activity of NAGase (a marker of cell damage) than that of S. uberis-infected EpH4-Ev cells without pretreatment (Fig. 5D). OXPHOS inhibition significantly increased TNF-α and IL-1β production (Fig. 5E-F), contrary to taurine pretreatment. Moreover, CPI-613 treatment significantly increased intracellular ROS levels (Fig. 5G) and NAGase activities (Fig. 5H) in EpH4-Ev cells during S. uberis infection. Inhibited glycolysis with 2-DG diminished the inflammatory response while CPI-613 treatment results in a sharp increase of inflammatory mediators and resultant cell damage in S. uberis infection. These results suggest that glycolysis and OXPHOS work differently in MECs challenged with S. uberis. Taurine decreases both glycolysis and OXPHOS balancing whole energy metabolism in S. uberis infection, relieving inflammation and protecting cells from OXPHOS breakdown thus attenuating a proinflammatory metabolic reaction.
Taurine regulates metabolic alterations in S. uberis infection by inhibiting the mTOR pathway
Previously, we show that mTOR was involved in the production of various inflammatory mediators during S. uberis infection (15). Enhancement of the glycolytic pathway correlates with mTOR activity (36). Therefore, we hypothesized that taurine coupled with cellular metabolism through the mTOR pathway optimizes the production of inflammatory biomolecules. Taurine pretreatment inhibited mTOR pathway activity in EpH4-Ev cells during challenge with S. uberis, as indicated by decreased mTOR phosphorylation and decreased phosphorylation of its downstream targets, p70 S6K and 4E-BP1 (Fig. 6A). The mTOR activator, MHY1485, reversed these changes in taurine-pretreated EpH4-Ev cells (Fig. 6A). To verify whether mTOR mediated energy metabolism during S. uberis infection, we assessed ECAR and OCR levels using MHY1485. ECAR and OCR decreased in taurine-pretreated EpH4-Ev cells exposed to S. uberis (Fig. 6B-C), whereas MHY1485 treatment increased these levels (Fig. 6B-C). The activities of key glycolysis-associated (HK, PFK, LDH) and OXPHOS-associated (PDH, SDH, mitochondrial complex IV) enzymes or components were consistent with the changes of ECAR and OCR (Fig. 6D-I). These findings confirm that taurine-mediated attenuation of metabolic responses in EpH4-Ev cells challenged with S. uberis is related to mTOR pathway activation. Additionally, TNF-α and IL-1β levels (Fig. 6J-K), ROS production (Fig. 6L and Fig. S8), and NAGase activity (Fig. 6M) significantly decreased in taurine-pretreated EpH4-Ev cells during S. uberis infection, whereas MHY1485 treatment exerted the opposite effects of taurine. These data establish that the mTOR signaling pathway mediates metabolic alterations of taurine in S. uberis infection.
Taurine rescues mTOR-mediated metabolic alterations in MECs via AMPK
As an AMPK activator (Fig. S9A), taurine has potential to negatively regulate mTOR activity resulting in coordination of cell metabolism with specific energy requirements (27). In EpH4-Ev cells, S. uberis infection increased the levels of phosphorylated AMPK versus controls (Fig. 7A). To determine whether the AMPK pathway was involved in the metabolic recovery induced by taurine, we assessed the expression status of the AMPK-mTOR-p70 S6K-4E-BP1 pathway in MECs after taurine pretreatment. Immunoblot analyses showed that taurine pretreatment significantly increased AMPK phosphorylation levels and subsequently downregulated mTOR, p70 S6K, and 4E-BP1 phosphorylation all of which function immediately downstream of mTOR (Fig. 7A). Pretreatment with the AMPK inhibitor, Compound C, blocked taurine-induced AMPK phosphorylation and reversed mTOR, p70 S6K, and 4E-BP1 phosphorylation (Fig. 7A). ECAR and OCR levels increased, which concurred with the activity level of the mTOR pathway (Fig. 7B-C). These data suggest that taurine-mediated regulation of metabolism in S. uberis-infected EpH4-Ev cells occurs via AMPK. Taurine significantly decreased HK, PFK, LDH, PDH, SDH, and mitochondrial complex IV activities in S. uberis infection, while pretreatment with Compound C reversed these taurine effects (Fig. 7D-I). Taurine significantly decreased the levels of TNF-α and IL-1β (Fig. 7J-K), ROS production (Fig. 7L and Fig. S9B), and NAGase activity (Fig. 7M) in S. uberis-infected EpH4-Ev cells, whereas Compound C pretreatment reversed these taurine-induced effects.