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Glutamine alleviates Staphylococcus aureus-induced mastitis by modulating macrophage polarisation [1]

['Chunhui Feng', 'Department Of Cardiology', 'China-Japan Union Hospital Of Jilin University', 'Changchun', 'Department Of Clinical Veterinary Medicine', 'College Of Veterinary Medicine', 'Jilin University', 'Jilin', 'Miao Liu', 'Zhaoqi He']

Date: 2026-03 

Abstract Mastitis, a major dairy industry challenge, is often caused by Staphylococcus aureus (S. aureus). Previous research linked gut microbiota disruption—induced by vancomycin in mice—to reduced microbial glutamine and increased mastitis susceptibility. This study hypothesizes that oral glutamine may regulate macrophage function and mitigate S. aureus mastitis. Our results showed that glutamine attenuated S. aureus-induced mastitis, protected the blood-milk barrier function, increased M2-type macrophages in the mammary gland, and increased the concentration of its metabolite α-Ketoglutaric acid (αKG) in both serum and mammary gland. In addition, the addition of αKG in mice significantly increased the transformation of M2-type macrophages and attenuated S. aureus-induced mastitis. Further studies revealed that αKG could initiate autophagy via oxidative phosphorylation, which in turn regulated the expression of Suppressor of cytokine signaling (SOCS3), a negative feedback inhibitor of the JAK/STAT signalling pathway. In vitro experiments verified that αKG enhances autophagy in macrophages, activates the JAK-STAT pathway, thereby promoting M2 polarization and alleviating S. aureus mastitis. This study revealed the key role of glutamine and its metabolite αKG in the mammary gland’s resistance to pathogenic infections, and reveal the mechanism by which they regulate macrophage polarization, providing theoretical guidance for solving the prevention and treatment problems of mastitis. This highlights a potential dietary intervention for mastitis management in dairy cows, bridging gut health and infection resistance.

Author summary Mastitis, a major challenge in the dairy industry, is commonly caused by S. aureus. This study demonstrates that oral glutamine supplementation alleviates S. aureus-induced mastitis in mice, protects the blood-milk barrier, and promotes an M2-polarized macrophage phenotype in mammary tissue. Its metabolite, α-ketoglutarate (αKG) enhances oxidative phosphorylation-driven autophagy, which downregulates SOCS3, a negative feedback inhibitor of the JAK-STAT pathway. This activation of JAK-STAT signaling promotes M2 macrophage polarization, thereby mitigating inflammation. In vitro experiments using RAW264.7 cells confirm that αKG enhances autophagy, activates JAK-STAT, and drives M2 polarization. These findings reveal a gut-mammary axis mediated by glutamine metabolism and highlight αKG as a critical immunometabolite that regulates macrophage function, offering a potential dietary strategy for mastitis prevention and therapy in dairy cows.

Citation: Feng C, Liu M, He Z, Liu Y, Yuan W, Xie P, et al. (2026) Glutamine alleviates Staphylococcus aureus-induced mastitis by modulating macrophage polarisation. PLoS Pathog 22(3): e1014053. https://doi.org/10.1371/journal.ppat.1014053 Editor: Rachel M. McLoughlin, Trinity College Dublin, IRELAND Received: September 3, 2025; Accepted: March 3, 2026; Published: March 11, 2026 Copyright: © 2026 Feng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The data that support the findings of this study are publicly available from manuscript and Supporting Information. Funding: The study is supported by the Jilin University’s Outstanding Young Talent Development Program (419250320048) to X.H. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Mastitis is the most common disease in dairy cows and is considered to be the most important disease threatening dairy production [1]. S. aureus is one of the most common pathogenic causes of mastitis in dairy cows [2]. Currently, the treatment of mastitis in dairy cows is still dominated by antibiotics [3]. In addition to the food safety problems caused by antibiotic abuse, its therapeutic effect has some limitations [4]. Therefore, finding effective new methods and approaches to prevent and treat mastitis is an urgent need for the current livestock breeding industry. Macrophages are an important part of the immune system and play vital roles in host defense and inflammation. Under different pathological conditions, macrophages are polarized into two inflammatory phenotypes: the proinflammatory M1 phenotype and the anti-inflammatory M2 phenotype [5]. The M1 phenotype consists of classically activated macrophages induced by various proinflammatory factors such as lipopolysaccharide (LPS), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) [6]. Conversely, M2 macrophages are alternatively activated macrophages induced by anti-inflammatory factors such as interleukin-4 (IL-4), glucocorticoids, and granulocyte colony-stimulating factor (G-CSF) [7]. The polarization between M1 and M2 macrophage phenotypes is a dynamic process that depends on the microenvironment and is regulated by multiple signals. Macrophages are characterized by heterogeneity and plasticity, and they exhibit different functions due to signals within the local microenvironment [8]. As central regulators of the innate immune system, their functional polarization directly determines the progression and outcome of inflammation. Numerous studies have shown that during the progression of mastitis, M1-like polarization of macrophages promotes the inflammatory response and exacerbates disease progression, whereas M2-like polarization suppresses the inflammatory response and reduces disease severity [9]. Although we refer to the M1/M2 phenotypes throughout the manuscript, this dichotomous description does not accurately reflect the multifunctional nature of macrophages in vivo. Previous studies have demonstrated that in addition to the exogenous pathogenic infections, there are also endogenous pathogenic pathways mediated by the gut microbiota and their metabolites that can lead to mastitis [10,11]. Furthermore, a study on the targeted clearance of gut Bacteroides found that the use of vancomycin significantly reduced the concentration of the glutamine. This concurrently increases susceptibility to S. aureus-induced mastitis. Metabolic alterations induced by the gut microbiota are the most direct way to mediate host-microbiota interactions and influence disease progression [12]. For example, deoxycholic acid (DCA) has been shown to inhibit S. aureus-induced activation of NF-κB and NLRP3 signalling in mouse mammary epithelial cells[11]. In type 2 diabetes, reductions in Roseobacter and Clostridium platensis result in reduced branched-chain amino acid transport and butyric acid biosynthesis, exhibiting increased insulin resistance [13]. Therefore, in the present study, we investigated whether glutamine exerts a protective effect against S. aureus-induced mastitis. Glutamine is the most abundant and versatile amino acid in the body, and it is plays a crucial role in the regulation of cellular signalling pathways, particularly those associated with the expression of genes involved in the regulation of inflammation and immune responses [14]. In virtually every cell, glutamine is used as a substrate for nucleotide synthesis (purines, pyrimidines, and aminoglycans), nicotinamide adenine dinucleotide phosphate (NADPH), antioxidants, and many other biosynthetic pathways involved in the maintenance of cellular integrity and function [15,16]. Some bovine studies have already demonstrated that glutamine supplementation affects the immune system by influencing both humoral and cellular immune responses and modulating the concentration of cytokines, thereby contributing to improved milk quality [17]. For example, glutamine pretreatment of bovine mammary epithelial cells inhibited the down-regulation of cellular expression of anti-oxidative stress factors and reduced the expression of proteins associated with induced inflammation [18,19]. According to previous research, α-ketoglutaric acid (αKG) plays a crucial role in immune regulation. For instance, in T cells and macrophages, it has been established that αKG modulates immune function by influencing epigenetic modifications and cellular metabolism [20,21]. Therefore, in this study, glutamine and its metabolite αKG were administered orally to mice to explore their effects and mechanisms on the S. aureus-induced mastitis.

Discussion Mastitis in dairy cows has a high incidence and prevalence rate, making it one of the most serious diseases currently threatening the dairy industry [26]. S. aureus is one of the most common pathogens causing mastitis, and antibiotic treatment is considered the most effective and widely used treatment method at present [27]. However, the overuse of antibiotics has led to increased production costs and drug residues, prompting pathogens to develop resistance [28,29]. Our previous research found that antibiotic treatment disrupted the gut microbiota in mice, resulting in increased permeability of the blood-milk barrier, enhanced migration of inflammatory cells across the barrier into the mammary gland, and ultimately the induction of mastitis [30]. Further study has demonstrated that inducing gut microbiota imbalance via vancomycin administration in drinking water increases the susceptibility of mice to S. aureus-induced mastitis. Concomitantly, the fecal concentration of the metabolite indole-3-propionic acid (IPA) was elevated, while that of glutamine was significantly reduced. in the feces significantly decreased. IPA is more indicative of a concomitant state of intestinal ecological imbalance or tissue inflammation. Notably, the levels of glutamine varied substantially among mice in the control group, which may reflect inherent physiological differences between individual animals. Nevertheless, the overall downward trend of glutamine levels in the vancomycin-treated group remained clear and consistent. Glutamine is widely recognized as a “fuel and signaling molecule” for immune cells, and it serves as a key amino acid for the proliferation and functional maintenance of lymphocytes and macrophages [31]. Previous studies have suggested that glutamine has a protective effect against mastitis, but the underlying mechanisms remain unclear [18,32]. Therefore, we investigated the role and mechanism of glutamine in S. aureus mastitis. Our results showed that glutamine protects the integrity of the blood-milk barrier, inhibits the inflammatory response, and suppresses the activation of the NF-κB signaling pathway induced by S. aureus. Glutamine is deaminated by glutaminase to form glutamic acid, which is then converted by glutamate dehydrogenase (GLUD) into αKG, which directly enters the TCA cycle [33]. Studies have demonstrated that glutamine exerts its immunomodulatory and anti-inflammatory effects through αKG. Specifically, α-KG supplementation reduced the proportion of CD45+F4/80+CD11b/c+TNFα+ ileal macrophages induced by glutamine treatment during necrotizing enterocolitis [34]. Additionally, other study has suggested that glutamine metabolism via the GLS1-AT-αKG pathway is a critical driver of vascular smooth muscle cell activation and survival [35]. In the present study, BPTES (a GLS 1 inhibitor) abolished the anti-inflammatory effects of glutamine, whereas αKG supplementation restored the protective effects against S. aureus-induced mastitis. These results indicate that αKG is a key signaling molecule for glutamine in the prevention of mastitis. Macrophages are believed to participate in the M1 phenotype during the early stages of infection, coordinating the host immune response, and subsequently acquire the M2 phenotype to suppress proinflammatory reactions and repair damaged tissues [36,37]. In the present study, we found that administration of αKG increased the number of M2-type macrophages, while treatment of cells with BPTES suppressed the expression of M2-specific genes, reduced αKG activity, and enhanced the inflammatory response in S. aureus-stimulated RAW 264.7 cells. However, addition of αKG reversed these changes. αKG supports macrophage proliferation and TCA cycle integrity, it provides energy support for M2 macrophages by promoting metabolic reprogramming such as fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) [38,39]. OXPHOS is a core process in cellular energy metabolism, generating ATP through the electron transport chain (ETC) and ATP synthase [40]. NADH and FADH₂ produced in the TCA cycle generate ATP through the ETC and produce ROS as by-products [41,42]. Moderate ROS can maintain metabolic homeostasis while inducing protective autophagy [43,44]. Results indicate that increasing autophagy activity may be beneficial during inflammation [45–47]. We found that αKG treatment increased the ROS level, ATP content, and OCR of the mammary glands of mice. In addition, the expression of autophagy markers ATG5 and LC-3Ⅱ was upregulated, while the expression of p62 was downregulated. Furthermore, oligomycin A abrogated the αKG-mediated enhancement of M2 polarization and the induction of inflammation in the in both in vivo and in vitro models of S. aureus-induced mastitis. Collectively, these results suggested that αKG regulates M2 polarization by modulating OXPHOS and autophagy. The autophagy adapter protein p62 recruits ubiquitinated SOCS3 to autophagosomes, which are subsequently degraded by lysosomes [48]. SOCS3 is a key negative feedback regulator of the JAK-STAT signaling pathway, particularly STAT3 [49,50]. SOCS3 binds to JAK1 via its SH2 domain, thereby blocking STAT3 phosphorylation, and competes with STAT3 for binding to the gp130 receptor, thereby reducing STAT3 activation [51]. In autophagy-deficient macrophages, SOCS3 overexpression inhibits the JAK-STAT pathway [52,53]. In the present study, we found that glutamine hydrolysis product αKG inhibits SOCS3 accumulation and activates the JAK/STAT pathway, which promotes M2 activation. This study utilized a mouse model of mastitis, in which immune responses, mammary gland anatomy, and metabolic characteristics differ from those of ruminants such as dairy cows. Further validation in cows or more physiologically relevant models is required in the future. The classic M1/M2 dichotomy was adopted to describe macrophage polarization in this study. Although this is a widely used theoretical framework, it does not fully reflect the potential continuum or mixed phenotypic states of macrophages in vivo. Future research could employ high-resolution techniques such as single-cell RNA sequencing to more precisely characterize the dynamic changes in macrophages during mastitis. Furthermore, this study did not fully rule out the potential influence of the mammary immune microenvironment on S. aureus itself.

Conclusion This study revealed that glutamine hydrolysis into αKG increases OXPHOS levels in vivo, promotes autophagy in tissue cells, inhibits the JAK/STAT pathway negative regulator SOCS3, and increases JAK/STAT pathway expression, thereby promoting macrophage M2 polarization, alleviating S. aureus-induced mastitis. It provides a theoretical basis for the development of mastitis prevention strategies for dairy cows, as well as the research and development of related supplements and functional feeds.

Materials and methods Ethics Statement All procedures involving animal experiments have been officially approved by the Animal Ethics Committee of Jilin University. Animals This study selected BALB/c female and male mice aged 8–12 weeks, and all experimental animals were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China). During the feeding process, we provided SPF-grade (pathogen-free) feed and drinking water for the mice. In terms of animal grouping and feeding management, we kept every two female mice and one male mouse together in the same cage until the female mice successfully become pregnant. Female mice were selected to construct an animal model of mastitis one week after delivery. In terms of experimental ethics and compliance, this study strictly adheres to the relevant provisions of the “Jilin University Guidelines for the Care and Use of Experimental Animals” to ensure that experimental operations comply with the highest ethical standards. Mouse mastitis model establishment and treatment The mouse mastitis model was induced by S. aureus. After the mice were anesthetized, the fourth mammary gland area of the mice was disinfected with 75% ethanol. The milk ducks were clamped and then 50 μL of S. aureus solution (10^6 CFU/mL) was injected into each nipple with a 100 μL syringe and left for 24 hours [54]. To verify the effect of glutamine on S. aureus-mastitis, 14 mice were divided into the glutamine group and the glutamine treatment mastitis group. Glutamine (G0200, Solarbio, China) was administered by gavage (50 mg/kg) to female mice 7 days before the establishment of the mastitis model. Furthermore, to verify whether αKG could inhibit S. aureus-induced mastitis, 14 female mice were divided into two groups (n = 7 per group). One week prior to mastitis model establishment, mice in the αKG treatment mastitis group received daily intraperitoneal injections of 10 mg/kg αKG (K1128, Sigma-Aldrich, USA) for 7 consecutive days [55], while mice in the αKG group received αKG only for the same duration. Subsequently, S. aureus mastitis was induced only in the αKG treatment mastitis group. After model establishment, mammary glands were collected for subsequent detection. Cell culture and treatment Mouse mononuclear macrophage leukaemia cells (RAW 264.7) were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. The cells were cultured in a humidified incubator at a temperature of 37°C and with 5% CO 2 . When the cell abundance reaches 90%, transfer the cells to a six-well plate for culture [56]. When the cell reached 90%. They were transferred to a six-well plate for culture and left to reach approximately 60% confluency. Media was then discarded and replaced with 2 mL of DEME medium containing 10% FBS to each well. In the S. aureus infection experiment, glutamine (4 mmol/L) and αKG (1 mmol/L) were added, respectively, to the DMEM medium (without glutamine) [57]. Oligomycin A of 1 mmol/L was added to detect the effect of oxidative phosphorylation levels on cell polarization. To detect the influence of the JAK1-STAT3 signaling pathway on cell polarization, 10 mmol/L Stattic (HY-13818, MedChemExpress, New Jersey, USA) was added to the culture medium. The treatment was carried out 12 h before the S. aureus infection. RAW 264.7 cells were cultured overnight in a six-well plate and then treated with S. aureus (105 CFU) for 4 h. Finally, the cells are collected for subsequent processing [58]. Haematoxylin-eosin (H&E) staining Mouse mammary samples used for histological analysis were fixed in 4% paraformaldehyde for 48 h. After paraffin embedding, 5 μm paraffin sections were prepared. After dewaxing and hydration, H&E staining was performed. The histological changes of the mammary gland were observed using an optical microscope. Based on the histological scoring system of mastitis research, the pathological damage level of each mammary was evaluated through three categories, including congestion/edema, lactation stasis/acinar necrosis, and neutrophil infiltration. Immunohistochemistry (IHC) Mammary gland specimens were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned at 5 μm thickness. Tissue sections were deparaffinized in xylene, rehydrated through a graded ethanol series, and subjected to antigen retrieval using citrate buffer (pH 6.0) or phosphate-buffered saline (PBS). Following blocking with goat serum at 37°C using a ready-to-use immunohistochemical kit (Kit-9710, Maxin Biotechnologies), sections were incubated overnight at 4°C with primary antibodies against ZO-1, Occludin, and Claudin-3. The sections were then treated with HRP-conjugated secondary antibodies, developed with 3,3’-diaminobenzidine (DAB-0031, Maxin Biotechnologies), and counterstained with hematoxylin. Finally, the stained sections were mounted with neutral resin and examined under a bright-field microscope for observation and imaging. Myeloperoxidase (MPO) activity determination The MPO Assay Kit (A044-1–1, Nanjing Institute of Bioengineering, China) was utilized to determine MPO activity in mammary gland tissue. Following the manufacturer’s protocol, the tissue was homogenized with the provided reagents, and the enzymatic activity of MPO was then quantified. Proinflammatory cytokines determination A 0.2 g sample of mammary gland tissue was precisely weighed and homogenized in 200 μL of PBS (pH = 7.4). After centrifugation at 12,000 rpm for 10 min (4 °C), the supernatant was collected. Subsequently, the concentrations of the proinflammatory cytokines IL-1β and TNF-α were measured using ELISA kits (Biolegend, CA, USA; Cat# 432604 and 430904) following the manufacturer’s guidelines. Western blot Mammary tissues and RAW 264.7 cells were homogenized with protein lysis buffer, and the total protein concentration in the homogenate supernatant was determined using the BCA protein quantification kit. In SDS-12% polyacrylamide gel, samples of different molecular weight proteins are separated and transferred to the PVDF membrane. After blocking with 5% skimmed milk at room temperature for three hours, the primary antibody was added to the membrane and incubated overnight at 4 °C. The membrane was then washed thrice with Tris Buffered Saline containing Tween 20 (X%), incubated with the secondary antibody for 2 h, and imaged using the Western blot detection system (Tanon 4500, Shanghai, China) with Chemistar High-sig ECl Western Blotting substrates (Tanon Technology, Shanghai, China). Protein bands were analysed using ImageJ software. β-actin was used as the internal reference to detect proteins on the membrane. The specific primary antibodies used in the experiment included p65 (#8242, Cell Signaling Technology, USA), p-p65 (#3033, Cell Signaling Technology, America), IκB (AF5002, Affinity Biosciences, Jiangsu, China), p-IκB (AF2002, Affinity Biosciences, Jiangsu, China), ZO-1 (AF5145, Affinity Biosciences, Jiangsu, China), Occludin (DF7504, Affinity Biosciences, Jiangsu, China), Claudin-3 (AF0129, Affinity Biosciences, Jiangsu, China), ATG5 (bsm-52596R, Bioss, Woburn, MA, USA), LC3-Ⅰ/LC3-Ⅱ (AF5402, Affinity Biosciences, Jiangsu, China), p62 (ab91526, Abcam plc, Cam bridge, UK), SOCS3 (bs-0580R, Bioss, Woburn, MA, USA), JAK1 (bs-1439R, Bioss, Woburn, MA, USA), p-JAK1 (bs-3238R, Bioss, Woburn, MA, USA), STAT3 (AF6294, Affinity Biosciences, Jiangsu, China), p-STAT3 (AF3293, Affinity Biosciences, Jiangsu, China) and β-actin (T0022, Affinity Biosciences, Jiangsu, China). αKG concentration determination The levels of αKG in mouse mammary tissue and serum were detected using the Amplex Red α-Ketoglutarate Assay Kit (Beyotime Biotechnology, Shanghai, China). According to the instructions provided by the reagent supplier, the collected mammary tissue was homogenized and centrifuged. The supernatant was aspirated to obtain the tissue sample. The tissue sample and serum were mixed with the working solution and added a 96-well plate. Absorbance was measured with a microplate reader at 570 nm, and the concentration of αKG in the mammary gland and serum calculated as per the suppliers’s instructions. Total RNA extraction and quantitative RT-PCR Total RNA was isolated from mouse mammary gland tissues and RAW 264.7 cells using TRIzol reagent (Life Technologies). For tissue samples, frozen tissues were pulverized in liquid nitrogen and homogenized in TRIzol. Cultured cells in 6-well plates were directly lysed with 1 mL TRIzol per well. Following chloroform phase separation, RNA was precipitated with isopropanol, washed with 75% ethanol, and finally dissolved in 15 μL DEPC-treated water under RNase-free conditions. For cDNA synthesis, 1 μg of total RNA was reverse transcribed using RT Master Mix for qPCR with gDNA Remover (ABclonal, Wuhan, China). Quantitative real-time PCR (qRT-PCR) was performed in triplicate using SYBR Green PCR Master Mix (KAPA Biosystems) on a StepOnePlus system (Applied Biosystems, CA, USA) according to the manufacturer’s protocol. Gene expression levels were calculated using the ΔΔCt method with BACTIN as the endogenous control (primer sequences are listed in Table 1). PPT PowerPoint slide

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TIFF original image Download: Table 1. Information for quantitative PCR primers. https://doi.org/10.1371/journal.ppat.1014053.t001 ROS determination The mouse mammary gland tissue was prepared into frozen sections. 5 μmol/l MitoSOX Red (MedChemExpress, New Jersey, USA) was added to completely cover the tissue. It was incubated at room temperature for 30 minutes and washed twice with PBS for 5 minutes each time. Observed with a confocal microscope, the Emission wavelength was 580 nm and the Excitation wavelength was 510 nm. ATP determination To determine the ATP content in mouse mammary tissue, the ATP assay kit (A095-1–1, Nanjing Institute of Bioengineering, China) was used. We weighed 0.02 g of the tissue, added 9 times the volume of cold double-distilled water, and homogenized it in an ice water bath to make a 10% homogenate. Then, we placed it in a boiling water bath, boiled it for 10 min, and centrifuged it at 3500 rpm for 10 min. We took the supernatant to obtain a mammary tissue sample. The ATP content was detected according to the instructions provided by the reagent supplier, and the colorimetric determination wavelength was 636 nm. Oxygen Consumption Rate (OCR) determination The Oxygen Consumption Assay Kit (BB-48211, Bestbio, Shanghai, China) was used to detect the oxygen consumption rate of cells. RAW 264.7 cell was cultured in 96-well plates. The culture medium was removed before detection and washed twice with PBS. Added 100 μL of oxygen blocking solution, placed the culture plate in the microplate reader (37 °C), and let it stand for 5 min before starting the detection. Excitation wavelength: 468 nm, emission wavelength: 603 nm. The OCR value was detected in real time by using a fluorescence microplate reader. Data was read every 10 min, and the total detection time was 120 min. Statistical analysis Data were analyzed using GraphPad Prism v8.0 (GraphPad Software, USA). Intergroup comparisons were conducted using an unpaired Student’s t-test, with results presented as mean ± standard error of the mean (SEM). Statistical significance was defined as P < 0.05. Metabolomics data analysis methods have been detailed in the preceding section.

Supporting information S1 Fig. Vancomycin-induced gut microbiota dysbiosis led to alterations in fecal metabolites, particularly glutamine concentration. Fecal metabolomic sequencing was conducted on samples obtained from both the control and vancomycin-treated groups. A: Box plot displaying the comparative concentrations of fecal amino acids and organic acids across the experimental groups. https://doi.org/10.1371/journal.ppat.1014053.s001 (DOCX) S2 Fig. α-KG elevates mitochondrial OXPHOS in mammary gland tissue, thereby inducing a higher level of autophagy. To determine whether inhibiting mitochondrial respiration reduces autophagy-related markers under these conditions, we conducted supplementary in vivo and in vitro assays. A: To further demonstrate the increase in autophagy within macrophages, Western blot analysis of autophagy markers was performed in RAW 264.7 cells. B: Following OXPHOS inhibition, the expression of autophagy markers in mammary gland tissue was evaluated upon supplementation with α-KG during S. aureus infection. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate significance from each group. ns, no significance. https://doi.org/10.1371/journal.ppat.1014053.s002 (DOCX) S1 Data. Box plot and original images of western blots. https://doi.org/10.1371/journal.ppat.1014053.s003 (RAR)

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