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. 2026 Jan 17;46(1):2615759. doi: 10.1080/01652176.2026.2615759

An underlying mechanism of bovine mastitis: PGE2 regulates Staphylococcus aureus-induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

Zhiguo Gong a,b,#, Zhuoya Yu a,b,#, Peipei Ren a,b, Shuangyi Zhang a,b, Ruifeng Gao a,b, Jiamin Zhao a,b, Yixin Wang a,b, Shaojie Qin a,b, Wenhui Bao a,b, Feng Shuang a,c,✉
PMCID: PMC12818336  PMID: 41546499

Abstract

Staphylococcus aureus (S. aureus) evades host immunity by modulating macrophage functions, including immune regulation and phagocytosis, ultimately contributing to bovine mastitis. This study aimed to elucidate the molecular mechanisms of S. aureus-induced bovine mastitis from both host and pathogen perspectives, focusing on prostaglandin E2 (PGE2) as a key regulator. During bovine mastitis, macrophages were recruited into the mammary gland with elevated inflammatory mediators. S. aureus lipoproteins amplified inflammation by activating MAPK and NF-κB pathways via TLR2, TLR4, and NLRP3, leading to elevated secretion of mediators, including PGE2, in bBMMs. Inhibition of TLR2, TLR4, or NLRP3 decreased COX-2 and mPGES-1 expression, suppressing PGE2 synthesis, while inhibition of COX-2 or mPGES-1 can regulate the expression of TLR2 and NLRP3, as well as the activation of MAPKs and NF-κB signaling pathways. Excess PGE2 can regulate inflammation and phagocytosis mediated by TLR2, TLR4, and NLRP3. S. aureus lipoproteins promote PGE2 synthesis via TLR2, TLR4, and NLRP3 signaling, while PGE2, in turn, modulates receptor activity, inflammation, and phagocytosis. These findings reveal crucial functional cross-talk between PGE2 and innate immune receptors in S. aureus-induced mastitis, suggesting that targeting this interaction may provide novel therapeutic strategies.

Keywords: Staphylococcus aureus, prostaglandin E2, pattern recognition receptors, cross-talk

1. Introduction

Bovine mastitis, primarily caused by Staphylococcus aureus (S. aureus), poses a significant challenge and substantial economic burden on the dairy industry (Li and Zhao 2018). The disease leads to severe economic losses, including decreased milk production, reproductive complications, culling of infected bovines, higher veterinary expenses, and the necessity to discard contaminated milk (Algharib et al. 2020). Additionally, alterations in milk composition, such as a reduction in β-lactoglobulin, may further compromise milk quality and immune regulation (Gao et al. 2022). Despite considerable progress in understanding S. aureus pathogenesis, how specific virulence-immune pathways shape eicosanoid production in the bovine mammary gland remains insufficiently defined, limiting targeted interventions for mastitis.

S. aureus infection-induced diseases are triggered by various virulence factors, including surface proteins, cell wall components, toxins, and enzymes (Ciloglu et al. 2021; Mohammad et al. 2022). The perfect combination of those virulence factors help S. aureus to adhere to host cells or tissues, resist phagocytes, lyse the leukocytes, evade immune killing, and finally cause systemic and focal infections in different organs (Ullah et al. 2021; Liao et al. 2023). Among the numerous virulence factors of S. aureus, a major class of surface proteins is lipoproteins (Lpps) (Mohammad et al. 2022). As major pathogen-associated molecular patterns (PAMP) of S. aureus, the maturation of Lpps is crucial for pathogenicity, inflammation, and immune signaling (Liao et al. 2023). Previous studies suggested that compared to the S. aureus SA113, the S. aureus SA113 isogenic mutant lgt::ermB (Δlgt), which is deficient in lipoprotein maturation, has a reduced ability to induce immune responses in monocytic, epithelial, and endothelial cells, resulting in decreased production of pro-inflammatory cytokines and chemokines (Stoll et al. 2005). These findings suggest that S. aureus virulence factors, particularly surface lipoproteins, are not only determinants of pathogenicity but also serve as key molecular signals that shape host immune recognition and response. During S. aureus infection in the host, Lpps structures act as ‘danger signals’ by activating pattern recognition receptors (PRRs), alerting the innate immune system to eliminate the potential pathogenic threat (Nguyen et al. 2017; Wu et al. 2020). PRRs are primarily expressed by innate immune cells such as monocytes, macrophages, neutrophils, and dendritic cells (Paerewijck and Lamkanfi 2022). In a healthy mammary gland, macrophages serve as critical sentinels, constantly surveilling for pathogens that may cause bovine mastitis (Pidwill et al. 2020; Imaizumi et al. 2024). Toll-like receptors (TLRs) are the primary receptors by which macrophages recognize PAMPs (Akira and Takeda 2004; Kawai and Akira 2010). TLRs play a significant role in bovine mastitis by mediating immune responses in the mammary gland (Islam et al. 2020). TLR2 is essential in recognizing pathogens like S. aureus, which are common culprits in mastitis (Liu et al. 2020). TLR2 helps detect microbial components such as lipoproteins from gram-positive bacteria, triggering inflammatory responses in the mammary epithelial cells (Sun et al. 2017). Furthermore, the NLR pyrin domain-containing 3 (NLRP3) inflammasome can be activated by pathogens such as S. aureus, leading to inflammation through pyroptosis in mammary epithelial cells (Yang et al. 2022; Wu et al. 2024). This activation often occurs via pathways like TLR2 and NF-κB, which play critical roles in orchestrating innate immune responses during mastitis (Wang et al. 2021; Wu et al. 2024). Furthermore, previous studies have shown that IL-1β-induced NF-κB activation downregulates miR-506 expression and exerts regulatory effects via JAG1, further underscoring the critical role of PRR/NF-κB signaling in inflammation (Hu et al. 2017).

Infected mammary tissues stimulate the release of arachidonic acid. This acid is then converted into prostaglandins through the cyclooxygenase (COX) pathway. (Atroshi et al. 1990; Caroline et al. 2009). This process is part of the body’s defense mechanism to control infection (Ryman et al. 2015). However, overproduction of prostaglandins can lead to excessive inflammation, resulting in tissue damage and prolonged healing times (Atroshi et al. 1990; Gruet et al. 2001; Chen H et al. 2024). Milk from mastitis quarters contains higher levels of leukotriene B4 and proteinoids (Arnardottir et al. 2016). This dual role of prostaglandins makes them both protective and potentially harmful in mastitis. The most dominant bioactive prostaglandins include PGD2, PGE2, and PGI2 (Ricciotti and FitzGerald 2011). PGE2, a substance produced during the inflammatory response, is synthesized from arachidonic acid (AA) present on the cell membrane, with Cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) playing a crucial role in this process (Ilari et al. 2020). Previous studies suggested that macrophages exposed to LPS upregulated the COX-2 enzyme, which leads to increased production of PGE2 (Janjic et al. 2018). PGE2 is the most abundant prostaglandin in mammals, synthesized in large amounts in response to cell-specific injury, stimulation, and pathogen infection (Shen et al. 2019). Research confirmed that activating the TLR2 signaling pathway promotes the secretion of PGE2, which is then involved in skin barrier repair (Fu et al. 2020; Liu et al. 2021; Serhan and Levy 2003). In addition, PGE2 can enhance the Pam3CSK4-induced inflammatory response by activating the TLR2/NF-κB signaling pathway in cells (Shen et al. 2019). S. aureus can promote PGE2 secretion by activating the TLR2, TLR4, and NLRP3 inflammasome signaling pathways in bovine neutrophils (Zhang et al. 2023). However, in bovine bBMMs, it remains unclear how S. aureus lipoproteins modulate the magnitude of inflammation and PGE2 biosynthesis/secretion, and how PGE2 in turn feeds back on TLR2, TLR4, and NLRP3 signaling.

Therefore, this study aimed to elucidate the molecular mechanisms underlying S. aureus lipoprotein-mediated regulation of PGE2 synthesis and macrophage function via TLR2, TLR4, and NLRP3 signaling in bovine mastitis, and to determine the feedback regulatory role of PGE2 in shaping innate immune responses. By delineating these pathways, this study aims to enable rational, mechanism-guided anti-inflammatory strategies for mastitis management.

2. Materials and methods

2.1. Ethics statement

All animal experiments were conducted following the regulations of the Chinese Experimental Animal Management Committee. The experimental protocol was approved by the Animal Welfare and Research Ethics Committee of Inner Mongolia Agricultural University (Approval ID: NND2022005).

2.2. Bacterial strains and animals

The following bacterial strains were employed in this study: (1) S. aureus SA113 wild-type (WT; ATCC 35556); (2) Lipoprotein maturation-deficient mutant (Δlgt, lgt::ermB); (3) Genetically complemented strain (+pRB; lgt::ermB + pRBlgt). These strains were kindly provided by Prof. Friedrich Götz from the Institute of Microbial Genetics, University of Tübingen, Germany (Stoll et al. 2005). To quantify bacterial load, 1 mL of S. aureus suspension (2 × 109 CFU) was cultured in 100 mL Mueller-Hinton II broth (BD Biosciences) at 37 °C with shaking for 16 h until reaching log-phase (OD600 ≈ 2.0). Serial dilutions were plated on MH agar and incubated at 37 °C for 16 h to determine CFU counts, consistently yielding approximately 2 × 109 CFU/mL across eight independent experiments.

Mammary tissue and rib samples were aseptically collected from licensed slaughterhouses, immediately placed in sterile containers, and transported under refrigeration to maintain tissue integrity for subsequent analyses.

2.3. S. aureus isolation and identification from the mammary tissue of bovine with mastitis

Mammary tissue samples from bovine with mastitis were swabbed with a sterile inoculation loop and inoculated onto S. aureus-selective agar medium, then incubated at 37 °C for 24 h. After incubation, colonies with typical S. aureus morphology were selected for Gram staining and microscopic examination. Selected colonies were then cultured in a liquid medium at 37 °C with shaking for sufficient bacterial growth. The genomic DNA of isolates was extracted by a genomic DNA purification kit (Promega Corporation, Madison, WI) according to manufacturer recommendations. The universal primers 27 F (forward primer 5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492 R (reverse primer 5′-GGTTACCTTGTTACGACTT-3′) were used to amplify the isolates’ 16S rRNA. The PCR product was sent for sequencing (Shanghai Shenggong Biology Engineering Technology Service, Ltd., China), and the results were analyzed using the NCBI BLAST algorithm for homologous sequence searches.

2.4. Immunohistochemistry and H&E staining

Mammary tissues were fixed using neutral formalin fixative for 24 h. Next, the tissues were dehydrated with graded ethanol, incubated with xylene, and embedded into paraffin. The paraffin blocks were cut into 4 μm sections, deparaffinized, rehydrated, and repaired using sodium citrate antigen retrieval solution. After the tissues were incubated with CD14 antibody (1: 100, Novus Biological, Littleton, CO, USA) overnight at 4 °C, they were washed with PBS and incubated with goat anti-rabbit IgG-HRP-conjugated secondary antibody (Abcam, Cambridge, UK) and diaminobenzidine (Solarbio, Beijing, China). After sealing the tissues with neutral gum (Solarbio, Beijing, China), a cell positive for CD14 was observed under an Axio Scan.Z1 slide scanner (Zeiss, Thornwood, NY).

2.5. Isolation and culture of bovine bone marrow-derived macrophages (bBMMs)

Bone marrow cells were isolated from rib segments (3 ± 0.2 cm) by PBS flushing, followed by filtration through a 40 μm cell strainer and centrifugation (1500 rpm, 8 min). After erythrocyte lysis (100 rpm, 5 min), cells were resuspended in RPMI 1640 medium supplemented with 20 ng/mL M-CSF and 20% FBS. To enrich for adherent macrophages, cells were subjected to five sequential 2-hour adherence steps in fresh culture flasks. The non-adherent cell fraction from the final flask was plated in 6-well plates (5 × 106 cells/well) and cultured for 6 days in M-CSF-containing medium with biweekly medium changes. Following differentiation, cells were maintained in 20% FBS/RPMI for 24 h. They were then polarized to M1 with 1 μg/mL LPS for subsequent experiments.

2.6. Experimental infections and treatment of bBMMs

The bBMMs (5 × 106 cells/well) were treated with the TLR2 inhibitor C29 (10−5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the TLR4 inhibitor TAK242 (10−5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the NLRP3 inhibitor MCC950 (10−5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 4 h before infection; the COX-2 inhibitor CAY10404 (10−5 M, Cayman Chemical Company, Ann Arbor, MI) was given for 40 min before infection, and the mPGES-1 inhibitor CAY10526 (10−5 M, Cayman Chemical Company, Ann Arbor, MI) was administered for 12 h before infection; the PGE2 (10−5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection. Then, cells were infected with S. aureus at multiplicities of infection (MOI) of 10:1 or 30:1. After 1 h incubation at 37 °C, extracellular bacteria were eliminated by gentamicin treatment (100 μg/mL). Cells were subsequently cultured until designated timepoints for analysis.

2.7. Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from S. aureus-infected bBMMs (4 h and 8 h post-infection) using TRIzol reagent (Invitrogen, Carlsbad, CA). Following DNase I treatment, cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA). Quantitative real-time PCR was performed using FastStart Universal SYBR Green Master Mix (Roche Applied Science, Mannheim, Germany) with gene-specific primers (Table 1) on an iCycler iQ5 system (Bio-Rad, Hercules, CA) under the following cycling conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Relative gene expression was normalized to Gapdh and calculated using the 2-ΔΔCt method, where ΔΔCt = ΔCt - ΔCt control and ΔCt = Ct (Target gene expression) - Ct (Gapdh gene expression).

Table 1.

Primers used for gene amplification, sequencing, and accession no.

Gene symbol Accession No. Primer sequence
β-actin NM_173979.3 Forward 5′-TCACCAACTGGGACGACA-3′
    Reverse 5′-GCATACAGGGACAGCACA-3′
TLR2 NM_174197.2 Forward 5′-CGATGACTACCGCTGTGACTC-3′
    Reverse 5′-CCTTCCTGGGCTTCCTCTT-3′
TLR4 NM_174198.6 Forward:5′-TGCCTTCACTACAGGGACTTT-3′
    Reverse:5′-TGGGACACCACGACAATAAC-3′
NLRP3 NM_001102219.1 Forward:5′-CAGATGAGCAGCAAGCAAGG-3′
    Reverse:5′ -ACAATCCAGCAGACCAGAGG-3′
COX-2 XM_007115297.3 Forward:5′-GGTGCCTGGTCTGATGATGT-3′
    Reverse:5′ -GATTAGCCTGCTTGTCTGGAAC-3′
mPGES-1 XM_027556544.1 Forward:5′-ATGGTACACACCGTGGCATA-3′
    Reverse:5′ -CACAATCTCAAAGGGCCATC-3′
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2.8. Western blot analysis

For protein extraction and western blot analysis, macrophages were lysed using M-PER mammalian protein extraction reagent (Thermo Scientific, Waltham, MA), with protein concentrations determined by BCA assay. Equal amounts of protein (10 μg/lane) were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with StartingBlock (TBS) Buffer (Thermo Scientific, Waltham, MA) for 1 h at 25 °C, membranes were incubated overnight at 4 °C with primary antibodies against phospho-ERK, ERK, phospho-p38, p38, phospho-NF-κB p65, NF-κB p65 (1:1000, Cell Signaling Technology, Beverly, MA); mPGES-1, COX-2 (1:1000), and GAPDH (1:10000, Abcam, Cambridge, UK). Following incubation with HRP-conjugated secondary antibodies (1:10000, Abcam, Cambridge, UK), protein bands were visualized using SuperSignal West Femto substrate (Thermo Scientific, Waltham, MA) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD). The antibodies used are listed in Table 2.

Table 2.

Antibodies used in Western blot.

Name Description KDa Concentration Company Cat No.
Phospho-p44/42 MAPK (Erk1/2) Rabbit monoclonal 44, 42 kDa 1:1000 Cell signaling Technology 4370T
p44/42 MAPK (Erk1/2) Rabbit monoclonal 44, 42 kDa 1:1000 Cell signaling Technology 4695T
Phospho-p38 MAPK (Thr180/Tyr182) Rabbit monoclonal 43 kDa 1:1000 Cell signaling Technology 4511T
p38 MAPK Rabbit monoclonal 40 kDa 1:1000 Cell signaling Technology 8690T
Phospho-NF-kB p65 (Ser536) Rabbit polyclonal 65 kDa 1:1000 Affinity Biosciences AF2006
NF-κB p65 Rabbit Monoclonal 65kDa 1:1000 Cell signaling Technology 4764T
Anti-Prostaglandin E Synthase/MPGES-1 Rabbit polyclonal 17 kDa 1:500 abcam ab62050
Cyclooxygenase-2/COX-2 Rabbit polyclonal 80 kDa 1:1000 Affinity Biosciences AF7003
GAPDH Rabbit monoclonal 36 kDa 1:10000 abcam ab4181603
Goat Anti-Ribbit IgG (H + L) HRP / 1:5000 Affinity Biosciences S0001
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2.9. Enzyme-linked immunosorbent assay (ELISA)

The collected mammary tissue was cut into smaller sections and weighed. Next, the samples were homogenized and lysed using T-PER Tissue Protein Extraction Reagent (Thermo Scientific, Waltham, MA). The lysates were centrifuged at 1,500 × g for 20 min at 4 °C. Concurrently, supernatants from bBMM cultures were collected, centrifuged (300 × g, 8 min, 4 °C) to remove cellular debris, and stored at −80 °C. Cytokine and chemokine levels (TNF-α, IL-1β, IL-10, PGE2) in both tissue lysates and cell culture supernatants were quantified using manufacturer-validated ELISA kits (TNF-α: R&D Systems, California, US; IL-1β/IL-10: Kingfisher, Saint Paul, USA; PGE2: Cayman Chemical Company, Ann Arbor, MI), with all assays performed in triplicate to ensure reproducibility.

2.10. Microscopy assay of bacterial phagocytosis

To assess PGE2’s role in phagocytosis, bBMMs were plated in 35 mm glass-bottom dishes (2 × 106 cells/well) and cultured for 24 h. Cells were pretreated for 24 h with either the mPGES-1 inhibitor CAY10526 (10−5 M), exogenous PGE2 (10−5 M), or left untreated as control. Following membrane labeling with 8 μM DiI fluorescent dye (30 min incubation in dark), cells were infected with Hoechst 33258-labeled S. aureus SA113 for 3 h before fixation with 4% paraformaldehyde. Phagocytic activity was assessed using a Zeiss LSM 800 confocal microscope (200× magnification) under standardized imaging conditions, with fluorescence intensity analysis performed to evaluate bacterial internalization. All experiments included at least three biological replicates to ensure data reproducibility.

2.11. Data analysis

All data were analyzed using GraphPad Prism 8 (GraphPad InStat Software, San Diego, CA) and are expressed as the mean ± standard deviation (SD). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s multiple comparisons test or two-way ANOVA with Bonferroni’s post-test, as appropriate. Differences with P values ≤ 0.05 were considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

3. Result

3.1. Pathological changes in mammary gland tissues and isolation and identification of pathogenic bacteria in bovine mastitis

We observed extensive purulent discharge on cross-sections from the mammary tissue collected at slaughter from bovine with mastitis. HE staining revealed significant infiltration of neutrophils and macrophages in the mammary ducts and stroma. Immunohistochemistry results showed that the CD14 expression increased in the bovine’s mammary tissue with mastitis (Figure 1A). Furthermore, TNF-α, IL-1β, IL-10, and PGE2 secretion levels were significantly higher in the mammary tissue of diseased bovine than that of healthy bovine (p < 0.05, Figure 1B). Pathogenic bacteria from the infected mammary tissue were isolated and identified through selective culturing on mannitol salt agar and Gram staining. The results indicated the presence of gram-positive cocci (Figure 1C). Additionally, a taxonomic analysis of the top 101 matches from the 16S rRNA sequencing results (shown in Supplementary Material 1) in comparison with NCBI data identified 3 sequences as Staphylococcus argenteus, 97 as Staphylococcus aureus, and 1 as Staphylococcus singaporensis (Table 3). Therefore, the isolated strains show 96% homology with Staphylococcus aureus.

Figure 1.

Figure 1.

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Pathological changes in mammary gland tissues and isolation and identification of pathogenic bacteria in bovine mastitis. The bovine mammary tissue with mastitis was collected from the slaughterhouse. Macroscopic analysis of pathological changes in the mammary tissue was performed, followed by hematoxylin and eosin (H&E) staining to observe microscopic pathological changes. Infiltration of macrophages was identified using immunohistochemistry with CD14 as a macrophage marker (A). Expression levels of TNF-α, IL-1β, IL-10 and PGE2 in the mammary tissue were quantified by ELISA (B). Detection of S. aureus in mammary tissue was performed by Gram staining and further confirmed through 16S rRNA sequencing (C). Results are expressed as mean ± SD of three independent experiments and were analyzed using one-way ANOVA with Bonferroni’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

Table 3.

Taxonomic attribution was performed using BLAST in the NCBI database.

Taxonomy Number of hits Number of organisms Description
Staphylococcus 101 3  
Staphylococcus argenteus 3 1 Staphylococcus argenteus hits
Staphylococcus aureus 97 1 Staphylococcus aureus hits
Staphylococcus singaporensis 1 1 Staphylococcus singaporensis hits
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3.2. Pathogen perspective: lipoproteins are essential for the S. aureus-induced inflammatory response in bBMMs

From the above results, we found that during mastitis in bovines, macrophages were heavily recruited to the site of inflammation as part of the body’s natural immune response to infection and release various inflammatory mediators. Activated macrophages play crucial roles in host defenses against S. aureus infection by initiating phagocytic activities and promoting inflammatory responses by producing various mediators (Pidwill et al. 2020; Ruslan 2008); however, the specific roles of S. aureus lipoproteins in S. aureus infection-induced inflammatory response in macrophages remain unclear. Thus, to analyze the effects of S. aureus lipoproteins on the secretion of inflammatory mediators, activation of the signaling pathway, and expression of TLR2, TLR4 and NLRP3, we used ELISA to detect the levels of inflammatory mediators, western blotting to measure the activation levels of signaling pathways, and qRT-PCR to analyze the expression of TLR2, TLR4, and NLRP3 in extracts from bBMMs infected with different S. aureus strains (SA113, Δlgt, and + pRB). The results showed that compared to bBMMs infected with SA113 S. aureus, the secretion of TNF-α, IL-1β, and IL-10 was significantly decreased in bBMMs infected with the Δlgt S. aureus strain (Figure 2A–C, p < 0.01). Furthermore, compared to the Δlgt S. aureus infection, the secretion of TNF-α, IL-1β, and IL-10 was significantly increased in bBMMs infected with the + pRB S. aureus strain (Figure 2A–C, p < 0.001). Previous studies suggested that the activation of the MAPKs and NF-κB signaling pathways promotes inflammation by inducing the expression of inflammatory chemokines and cytokines (Zhang et al. 2022). Further, we measured the activation of MAPKs and NF-κB signaling pathways. The results showed that compared to the SA113 S. aureus infection, the phosphorylation levels of p65, p38, and ERK were significantly attenuated in bBMMs infected with the Δlgt S. aureus strain (Figure 2D, p < 0.05). Furthermore, the + pRB S. aureus infection caused higher phosphorylation levels of p65, p38, and ERK in bBMMs compared to the Δlgt S. aureus infection (Figure 2E–G, p < 0.05).

Figure 2.

Figure 2.

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Pathogen perspective: Lipoproteins are essential for the S. aureus-induced inflammatory response in bBMMs. The bBMMs were infected with S. aureus SA113 (WT), isogenic mutant lgt::ermB (Δlgt), or its complemented strain, lgt::ermB + pRBlgt (+pRB), at MOI 10:1 or not infected. The secretion of TNF-α, IL-1β and IL-10 were detected by ELISA (6, 9, and 12 h after infection) (A–C). The activation of the MAPK (P-ERK and P-p38) and NF-κB (P-p65) pathways was evaluated by western blotting at 15, 30, and 60 min post-infection (D). TLR2, TLR4 and NLRP3 mRNA expression levels were detected by qRT-PCR and normalized to those of the housekeeping gene β-actin at 4 h post-infection (E–G). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post-hoc test. *P < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

Next, we analyzed the expression of TLR2, TLR4, and NLRP3. We found that compared to the SA113 S. aureus infection, the expression of TLR2, TLR4, and NLRP3 was significantly reduced in bBMMs infected with Δlgt S. aureus (Figure 2E–G, p < 0.01). Additionally, the + pRB S. aureus infection led to higher expression levels of TLR2, TLR4, and NLRP3 in bBMMs compared to the Δlgt S. aureus infection (Figure 2E–G, p < 0.05). These results demonstrate that during S. aureus infection, S. aureus lipoproteins enhance the inflammatory response by mediating TLR2, TLR4, and NLRP3, which in turn activate the MAPKs and NF-κB signaling pathways, leading to the secretion of inflammatory mediators in bBMMs.

3.3. Pathogen perspective: lipoproteins play essential roles in PGE2 production induced by S. aureus infection in bBMMs

PGE2 is largely synthesized in response to cell-specific trauma, stimuli, pathogen infection, or signaling molecules, making the control of PGE2 synthesis a crucial anti-inflammatory strategy (Serhan and Levy 2003; Vane and Botting 2003; Park et al. 2006). However, it is unknown whether lipoproteins can induce PGE2 production. The results showed that SA113 S. aureus infection led to higher protein expression levels of COX-2 and mPGES-1 in bBMMs compared to Δlgt S. aureus infection at the indicated time points (12 h or 24 h post-infection, p < 0.05, Figure 3A). Additionally, COX-2 mRNA and mPGES-1 mRNA expression increased in bBMMs infected with SA113 S. aureus and + pRB S. aureus for 4 h and/or 8 h (p < 0.0001) compared to those infected with Δlgt S. aureus (Figure 3B,C). In addition to the expression and synthesis of the enzymes COX-2 and mPGES-1 involved in PGE2 synthesis, we directly examined the secretion of PGE2. The results showed that compared to the SA113 S. aureus infection, the secretion of PGE2 was significantly attenuated in bBMMs infected with Δlgt S. aureus (Figure 3D, p < 0.0001). Furthermore, the + pRB S. aureus infection led to higher PGE2 secretion levels in bBMMs compared to Δlgt S. aureus infection (Figure 3D, p < 0.01). These results demonstrate that during S. aureus infection, S. aureus lipoproteins promote the production of PGE2 by inducing the expression of COX-2 and mPGES-1 in bBMMs.

Figure 3.

Figure 3.

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Pathogen perspective: Lipoproteins play essential roles in PGE2 production induced by S. aureus infection in bBMMs. The bBMMs were infected with S. aureus SA113 (WT), isogenic mutant lgt::ermB (Δlgt), or its complemented strain, lgt::ermB + pRBlgt (+pRB), at MOI 10:1 or not infected. The expedition of the COX-2 and mPGES-1 was evaluated by western blotting at 12 h and 24 h post-infection (A). COX-2 and mPGES-1 mRNA expression levels were detected by qRT-PCR and normalized to those of the housekeeping gene β-actin at the 4 h and 8 h post-infection (B, C). The secretion of PGE2 were detected by ELISA (9 h after infection). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

3.4. Host perspective: TLR2, TLR4, and NLRP3 play essential roles in the inflammatory response induced by S. aureus infection in bBMMs

Above, we analyzed the role of S. aureus lipoproteins from the pathogen’s perspective. In this part, we will explore the roles of TLR2, TLR4, and NLRP3 in the inflammatory response caused by S. aureus infection from the host’s perspective. The results showed that SA113 S. aureus infection induced high ERK, p38, and p65 phosphorylation levels compared to uninfected bBMMs (15, 30, and 60 min post-infection, p < 0.01, Figure 4A). Additionally, pretreatment with C29 (TLR2 inhibitor), TAK242 (TLR4 inhibitor), and MCC950 (NLRP3 inhibitor) downregulated the phosphorylation of ERK, p38, and p65 in SA113 S. aureus-infected bBMMs (p < 0.01, Figure 4A). Further research showed that pretreatment with C29, TAK242, and MCC950 downregulated the secretion of IL-1β at 4, 9, and 12 h post-infection with S. aureus, downregulated IL-10 secretion at 9 h post-infection, and downregulated TNF-α secretion at 12 h post-infection in bBMMs (p < 0.0001, Figure 4B–D). These results demonstrate that TLR2, TLR4, and NLRP3 play essential roles in S. aureus-infected bBMMs, and their activation can promote the MAPKs and NF-κB signaling pathways-mediated secretion of inflammatory mediators.

Figure 4.

Figure 4.

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Host perspective: TLR2, TLR4, and NLRP3 play essential roles in the inflammatory response induced by S. aureus infection in bBMMs. bBMMs were pretreated with the TLR2 inhibitor (C29, 10−5 M, before infection for 1 h), TLR4 inhibitor (TAK242, 10−5 M, before infection for 1 h), and NLRP3 inhibitor (MCC950, 10−5 M, before infection for 4 h). Then, bBMMs were infected SA113 (MOI 10:1), or uninfected. The activation of the MAPK (P-ERK and P-p38) and NF-κB (P-p65) pathways was evaluated by western blotting at 15, 30, and 60 min post-infection (A). The secretion of TNF-α, IL-1β and IL-10 were detected by ELISA (6, 9, and 12 h after infection) (B–D). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

3.5. Host perspective: TLR2, TLR4, and NLRP3 play essential roles in PGE2 production induced by S. aureus infection in bBMMs

This part studied the detailed roles of TLR2, TLR4, and NLRP3 in the synthesis and secretion of PGE2. After pretreatment with inhibitors (C29, TAK242, and MCC95), qRT-PCR and western blot were used to analyze the expression of COX-2 and mPGES-1, and ELISA was used to detect the secretion of PGE2 in S. aureus-infected bBMMs. The results showed that S. aureus infection could induce high levels of COX-2 and mPGES-1 expression, while pretreatment with C29, TAK242, and MCC95 could downregulate the expression of COX-2 and mPGES-1 in bBMMs (p < 0.001, Figure 5A). The qRT-PCR results were consistent with the western blot results, also showing that the inhibitors could downregulate the expression of COX-2 and mPGES-1 in S. aureus-infected bBMMs (p < 0.001, Figure 5B,C). In addition, S. aureus infection could induce high levels of PGE2 secretion, and pretreatment with C29, TAK242, and MCC95 could down-regulate PGE2 secretion in bBMMs (p < 0.0001, Figure 5D). These results demonstrate that TLR2, TLR4, and NLRP3 affect PGE2 synthesis and secretion in S. aureus-infected bBMMs via COX-2 and mPGES-1.

Figure 5.

Figure 5.

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Host perspective: TLR2, TLR4, and NLRP3 play essential roles in PGE2 production induced by S. aureus infection in bBMMs. bBMMs were pretreated with the TLR2 inhibitor (C29, 10−5 M, before infection for 1 h), TLR4 inhibitor (TAK242, 10−5 M, before infection for 1 h), and NLRP3 inhibitor (MCC950, 10−5 M, before infection for 4 h). Then, bBMMs were infected SA113 (MOI 10:1), or uninfected. The experession of the COX-2 and mPGES-1 was evaluated by western blotting at 12 h and 24 h post-infection (A). COX-2 and mPGES-1 mRNA expression levels were detected by qRT-PCR and normalized to those of the housekeeping gene β-actin at the 4 h and 8 h post-infection (B, C). The secretion of PGE2 were detected by ELISA (9 h after infection) (D). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

3.6. Cross-talk: PGE2 regulates TLR2, TLR4, and NLRP3 expression and inflammatory responses in S. aureus-infected bBMMs

The results of the above four sections analyze the effects of S. aureus lipoproteins on host inflammatory response and PGE2 synthesis and secretion from both pathogen and host perspectives, as well as the impact of the host PRRs (TLR2, TLR4 and NLRP3) on inflammation and PGE2 synthesis and secretion. Thus, this section analyzes the role of PGE2 in TLR2, TLR4, and NLRP3 expression and mediation of the inflammatory response in S. aureus-infected bBMMs. CAY10526 (mPGES-1 inhibitor) and CAY10404 (COX-2 inhibitor) were used to inhibit PGE2 synthesis and secretion in S. aureus-infected bBMMs. The results showed that CAY10526 and CAY10404 could down-regulate PGE2 synthesis and secretion in S. aureus-infected bBMMs (p < 0.0001, Figure 6A). Furthermore, S. aureus infection could induce high TLR2, TLR4, and NLRP3 expression levels. CAY10526 and CAY10404 could also downregulate the expression of TLR2 and NLRP3 (p < 0.05, Figure 6B,D). However, they did not affect TLR4 expression in bBMMs (Figure 6C). During S. aureus infection, TNF-α, IL-1β, and IL-10 secretion levels were high. CAY10526 and CAY10404 could attenuate IL-1β secretion in bBMMs (p < 0.05, Figure 6F,I). However, CAY10526 and CAY10404 pretreatment could increase IL-10 secretion in S. aureus-infected bBMMs (p < 0.0001, Figure 6G,J). CAY10526 and CAY10404 inhibited TNF-α secretion at different time points; CAY10526 inhibited TNF-α secretion at 12 h, while CAY10404 inhibited TNF-α secretion at 9 h in S. aureus-infected bBMMs (p < 0.05, Figure 6E,H). In addition to analyzing the effect of PGE2 on the host inflammatory response, the effect of PGE2 on intracellular killing was also examined. The results showed that during S. aureus infection, CAY10526 and CAY10404 did not affect intracellular killing (Figure 6K). These results demonstrate that PGE2 could regulate TLR2- and NLRP3-mediated inflammatory mediator production in S. aureus-infected bBMMs.

Figure 6.

Figure 6.

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Cross-talk: PGE2 regulates TLR2, TLR4, and NLRP3 expression and inflammatory responses in S. aureus-infected bBMMs. bBMMs were pretreated with the COX-2 inhibitor (CAY10404, 10−5 M, before infection for 40 min), mPGES-1 inhibitor (CAY10526, 10−5 M, before infection for 12 h). Then, bBMMs were infected SA113 (MOI 10:1), or uninfected. The secretion of PGE2 were detected by ELISA (9 h after infection) (A). TLR2, TLR4 and NLRP3 mRNA expression levels were detected by qRT-PCR and normalized to those of the housekeeping gene β-actin at 4 h post-infection (B–D). The secretion of TNF-α, IL-1β and IL-10 were detected by ELISA (9 h and 12 h after infection) (E–J). Phagocytosis of Hoechst 33258 (blue)-labelled SA113 S. aureus within DiI-labelled macrophages (Orange) was analyzed by microscopy assay (×400, K). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

3.7. Cross-talk: excess PGE2 exacerbates inflammation and impairs intracellular killing in S. aureus-infected bBMMs

Based on the above studies, we determined that blocking PGE2 synthesis could regulate the TLR2- and NLRP3-mediated inflammatory response in bBMMs. Thus, this section analyzed the effect of excess PGE2 (exogenous PGE2 pretreatment) on the inflammatory response and macrophage phagocytosis in S. aureus-infected bBMMs.The results showed that exogenous PGE2 pretreatment could up-regulate the expression of TLR2, TLR4, and NLRP3 in S. aureus-infected bBMMs (p < 0.01, Figure 7A–C). In addition, during S. aureus infection, compared with exogenous PGE2 pretreatment cells, the untreated cells showed lower levels of ERK and p65 phosphorylation (15, 30, and 60 min post-infection) (p < 0.05, Figure 7D). Exogenous PGE2 pretreatment cells showed higher p38 phosphorylation than PGE2 untreated cells in S. aureus-infected cells (15 min post-infection, p < 0.0001, Figure 7D). Exogenous PGE2 pretreatment could upregulate the secretion of TNF-α, IL-1β, and IL-10 in S. aureus-infected bBMMs (p < 0.01, Figure 7E–G). The results showed that compared to infection with Δlgt S. aureus, infection with Hoechst 33258-labelled SA113 S. aureus induced higher levels of intracellular killing in bBMMs (p < 0.0001, Figure 7H). PGE2 pretreatment decreased intracellular killing of SA113 S. aureus by bBMMs (p < 0.01, Figure 7H). These results demonstrate that excess PGE2 could regulate the inflammatory response and macrophage phagocytosis in S. aureus-infected bBMMs.

Figure 7.

Figure 7.

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Cross-talk: Excess PGE2 exacerbates inflammation and impairs intracellular killing in S. aureus-infected bBMMs. bBMMs were pretreated with the PGE2 (10−6 M, before infection for 24 h). Then, bBMMs were infected SA113 (MOI 10:1), or uninfected. TLR2, TLR4 and NLRP3 mRNA expression levels were detected by qRT-PCR and normalized to those of the housekeeping gene β-actin at 4 h post-infection (A–C). The activation of the MAPK (P-ERK and P-p38) and NF-κB (P-p65) pathways was evaluated by western blotting at 15, 30, and 60 min post-infection (D). The secretion of TNF-α, IL-1β and IL-10 were detected by ELISA (9 h and 12 h after infection) (E–G). Phagocytosis of Hoechst 33258 (blue)-labelled SA113 S. aureus within DiI-labelled macrophages (Orange) was analyzed by microscopy assay (×400, H). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

4. Discussion

Bovine mastitis is a prevalent issue that presents substantial economic challenges to the livestock industry (Algharib et al. 2020). Mastitis caused by S. aureus results in a significant drop in milk production and deterioration in milk quality and may necessitate the culling of affected animals (Campos et al. 2022). The treatment costs for bovine mastitis impose an additional financial strain on livestock operations. S. aureus is a common gram-positive pathogen that carries various virulence factors and can cause bovine mastitis (Zhao et al. 2024). Lipoprotein, as one of the classical PAMPs expressed by S. aureus, mainly induce the host to produce an innate immune response and related inflammatory response to cause the diseases (Mohammad et al. 2022). Macrophages are essential for innate immune responses and play crucial roles in phagocytosis and the activation of inflammation (Thomas et al. 2013). Previous studies showed that during S. aureus infection, lipoproteins could activate TLR2 and increase TLR2-associated inflammatory cytokines production in macrophages (Wu et al. 2020). In S. aureus-induced brain abscesses, the host’s defense against S. aureus infection depends not only on TLR2 but also requires TLR4 (Stenzel et al. 2008). Also, NLRP3 is crucial for macrophage IL-1β production in response to TLR agonists plus ATP and to gram-positive bacteria such as S. aureus (Martinon et al. 2009; Masters et al. 2009). NLRP3, which is essential for macrophage IL-1β production in response to TLR agonists and Gram-positive bacteria such as S. aureus, has also been implicated in the regulation of pyroptosis, as supported by recent evidence (Trinchieri and Sher 2007). Evidence suggests that NLRP3 regulates the progression of ovarian cancer through its modulation of pyroptosis (Liu et al. 2025). This leads to the activation of many intracellular signaling pathways, followed by the production of inflammatory mediators, among which PGE2 is a typical inflammatory mediator (Timothy et al. 2013; Benjamin et al. 2023). PGE2 is a major lipid produced by arachidonic acid (AA) metabolism. PGE2, a key mediator of inflammation, is significantly elevated in the milk of bovines with mastitis compared to healthy bovines, reflecting its role in the inflammatory response associated with the disease (Kuehl and Egan 1980; Atroshi et al. 1986). However, during S. aureus infection, the mechanisms by which PGE2 regulates the host inflammatory response and its interactions with TLR2, TLR4, and NLRP3 remain unclear. In this study, we first aimed to evaluate the effect of S. aureus lipoproteins on the inflammatory response and PGE2 synthesis and secretion in bBMMs infected with S. aureus. Our study found that macrophages are actively recruited to the inflammation site during bovine mastitis and release a range of inflammatory mediators (Figure 1A,B). It is worth noting that we isolated and cultured S. aureus from the mammary tissue of bovine with mastitis (Figure 1C). Previous studies showed the presence of viable S. aureus in macrophages in milk samples from animals with bovine mastitis (Cai et al. 2020). Study showed the dual RPA-LFIA enables rapid, sensitive, and efficient detection of S. aureus in milk through a single RPA reaction coupled with LFIA-based colorimetric readout (Zhang et al. 2025).

S. aureus possesses several virulence factors that help it evade immune responses and survive within host cells, particularly macrophages (Malak et al. 2020; Pidwill et al. 2020). Lipoproteins from S. aureus play a key role in immune evasion by manipulating immune signaling to prevent an effective immune response, which enables the bacteria to persist within immune cells like macrophages and evade host defenses (Muñoz-Planillo et al. 2009). The presence of S. aureus lipoproteins could increase the inflammatory response by promoting the expression of TLR2, TLR4, and NLRP3, activating MAPKs and NF-κB signaling pathways, and inducing inflammatory mediator secretion in S. aureus-infected bBMMs (Figure 2). Previous studies showed that S. aureus lipoproteins can interfere with mechanisms such as complement activation, which is crucial for opsonization and phagocytosis, and modulate autophagy pathways within bovine mammary epithelial cells, promoting intracellular survival of the bacteria (Geng et al. 2020). Additionally, lipoproteins in S. aureus contribute to its ability to adhere to the mammary epithelial cells, facilitating colonization and infection of the mammary gland, leading to mastitis (Neelam et al. 2022; Amanzholova et al. 2024). We found that S. aureus lipoproteins influence PGE2 secretion by affecting COX-2 and mPGES-1 expression (Figure 3). All these findings suggest that S. aureus lipoproteins are crucial immunobiologically active components that can cause mastitis.

Next, we aimed to evaluate the effect of TLR2, TLR4, and NLRP3 on the inflammatory response, and PGE2 synthesis and secretion in S. aureus-infected bBMMs. The deficiency of TLR2, TLR4, and NLRP3 decreases inflammatory mediator secretion by impairing the activation of MAPKs and NF-κB signaling pathways in S. aureus-infected bBMMs (Figure 4). A bacterial stimulus such as lipoprotein or lipopolysaccharide triggers a priming step through TLR2- or TLR4-mediated NF-κB signaling, resulting in the production of pro-interleukin IL-1β and pro-IL-18 and the transcription and post-translational modification of NLRP3 (Wang et al. 2020). Furthermore, TLR2, TLR4, and NLRP3 inhibitor treatment can reduce the expression of COX-2 and mPGES-1, further decreasing the secretion of PGE2 in S. aureus-infected bBMMs (Figure 5). Previous studies showed that S. aureus infection in bovines leads to increased PGE2 production in neutrophils, with TLR2 being one of the receptors mediating this process (Zhang et al. 2023). Previous studies have indicated that growth factors exert regulatory effects on immune responses, suggesting a close interplay between these pathways (Chen F et al. 2024). All these findings suggest that TLR2, TLR4, and NLRP3 play crucial roles in the inflammatory response, and PGE2 synthesis and secretion caused by S. aureus.

Bovine mastitis is usually accompanied by the up-regulation of COX-2 expression and the increased PGE2 production (Atroshi et al. 1986). In the above studies, we explored the influence of TLR2, TLR4 and NLRP3 on the synthesis and secretion of PGE2. In this part, we aimed to explore the role of PGE2 in TLR2, TLR4 and NLRP3 expression and its mediated inflammatory response. During S. aureus infection, COX-2 and mPGES-1 inhibitors could block the synthesis and secretion of PGE2. Additionally, inhibitor treatment could decrease the expression of TLR2 and NLRP3 in S. aureus-infected bBMMs (Figure 6). Notably, excess PGE2 treatment could increase the expression levels of TLR2, TLR4, and NLRP3, as well as the activation of NF-κB and MAPK in S. aureus-infected bBMMs (Figure 7). Previous studies showed that PGE2 could promote NLRP3 priming via the EP4-cAMP-PKA signaling pathway, leading to macrophage NF-κB activation (Jimenez-Duran and Triantafilou 2021). It is known that NLRP3 promotes the maturation of the precursor of IL-1β and the secretion of active IL-1β through the activation caspase-1 (Matthew et al. 2018). The secretion of IL-1β decreased after COX-2 and mPGES-1 inhibitor treatment in S. aureus-infected bBMMs (Figure 6). However, excess PGE2 treatment increased the secretion of IL-1β in S. aureus-infected bBMMs (Figure 7). This trend is consistent with the expression trend of NLRP3. This result further demonstrated that during S. aureus infection, PGE2 is involved in NLRP3-mediated IL-1β secretion.

In bovine mastitis, particularly those triggered by bacterial infections, macrophages recruit neutrophils and other immune cells to the infection site (Bianchi et al. 2019). Macrophages are essential to the innate immune response, eliminating pathogens via phagocytosis and initiating the adaptive immune response by producing cytokines and presenting antigens (Hind et al. 2015). We found that COX-2 and mPGES-1 inhibitors blocking the synthesis and secretion of PGE2 did not affect phagocytosis in S. aureus-infected bBMMs (Figure 6). However, excess PGE2 treatment reduced the phagocytosis of macrophages in S. aureus SA113-infected bBMMs (Figure 7). Other studies also have shown the participation of lipid inflammatory mediators, PGE2 in phagocyte antimicrobial effector functions upon infection of different pathogens (Araújo-Santos et al. 2014; Bonyek-Silva et al. 2020). Additionally, S. aureus lipoproteins also engaged in regulating the phagocytosis of macrophages (Figure 7). Interestingly, when S. aureus lipoproteins are deficient, excess PGE2 treatment does not affect the phagocytosis of macrophages (Figure 7). Previous studies indicate that Psa, a homopolymer macromolecular complex, inhibits phagocytosis by binding to bacterial lipoproteins, preventing host immune cells from recognizing the pathogen (Ke et al. 2013). However, the detailed mechanism of this discovery, the relationship of PGE2 and lipoproteins with phagocytosis, needs more research.

5. Conclusion

In conclusion, this study demonstrates that macrophages play a central regulatory role in Staphylococcus aureus-induced bovine mastitis by modulating the inflammatory response through intricate cross-talk among TLR2, TLR4, NLRP3, and PGE2 signaling pathways. Importantly, we show that PGE2 not only influences the onset and progression of inflammation but also affects macrophage phagocytic activity, a process that can be disrupted by S. aureus lipoproteins (Figure 8). These findings highlight a critical mechanism underlying host-pathogen interactions in bovine mastitis, offering valuable insights into potential therapeutic targets aimed at controlling inflammation and improving disease outcomes in dairy cattle.

Figure 8.

Figure 8.

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Graphical abstract of the present study: the involvement of TLR2-, TLR4-, and NLRP3-dependent PGE2 signaling in macrophage responses to S. aureus, which modulates inflammatory signaling and phagocytic activity and thereby contributes to the pathogenesis of bovine mastitis.

Supplementary Material

Supplementary Material 1.docx
TVEQ_A_2615759_SM6152.docx (14.9KB, docx)

Funding Statement

This work was supported by the National Natural Science Foundation of China (No. 32260903; 32202879), Allocation of research funds for universities directly affiliated with the Inner Mongolia Autonomous Region (grant number BR220125); Natural Science Foundation of Inner Mongolia of China (2024MS03064); Inner Mongolia Autonomous Region Graduate Research and Innovation Funding Program (S20231098Z).

Author contributions

Conceptualization: ZGG, ZYY, SF, SYZ. Data curation: ZYY, RFG, SF, PPR. Formal analysis: JMZ, YXW, SJQ. Funding acquisition: SF, SYZ. Investigation: WHB, RFG. Methodology: YXW, JMZ. Project administration: SF, SYZ. All authors have read and approved the final work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data for this study are available by contacting the corresponding authors upon reasonable request.

References

  1. Akira S, Takeda K.. 2004. Toll-like receptor signalling. Nat Rev Immunol. 4(7):499–511. 10.1038/nri1391 [DOI] [PubMed] [Google Scholar]
  2. Algharib SA, Dawood A, Xie S.. 2020. Nanoparticles for treatment of bovine Staphylococcus aureus mastitis. Drug Deliv. 27(1):292–308. 10.1080/10717544.2020.1724209 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amanzholova M, Shaizadinova A, Bulashev A, Abeldenov S.. 2024. Genetic identification of Staphylococcus aureus isolates from cultured milk samples of bovine mastitis using isothermal amplification with CRISPR/Cas12a-based molecular assay. Vet Res Commun. 48(1):291–300. 10.1007/s11259-023-10212-z [DOI] [PubMed] [Google Scholar]
  4. Araújo-Santos T, et al. 2014. Prostaglandin E2/leukotriene B4 balance induced by Lutzomyia longipalpis saliva favors Leishmania infantum infection. Parasit Vectors. 7(1):601. 10.1186/s13071-014-0601-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Arnardottir H, Orr SK, Dalli J, Serhan CN.. 2016. Human milk proresolving mediators stimulate resolution of acute inflammation. Mucosal Immunol. 9(3):757–766. 10.1038/mi.2015.99 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Atroshi F, Parantainen J, Sankari S, Osterman T.. 1986. Prostaglandins and glutathione peroxidase in bovine mastitis. Res Vet Sci. 40(3):361–366. 10.1016/S0034-5288(18)30551-4 [DOI] [PubMed] [Google Scholar]
  7. Atroshi F, Sankari S, Rizzo A, Westermarck T, Parantainen J.. 1990. Prostaglandins, glutathione metabolism, and lipid peroxidation in relation to inflammation in bovine mastitis. Adv Exp Med Biol. 264:203–207. 10.1007/978-1-4684-5730-8_33 [DOI] [PubMed] [Google Scholar]
  8. Benjamin PH, et al. 2023. Staphylococcus aureus host interactions and adaptation. Nat Rev Microbiol. 21(6):380–395. 10.1038/s41579-023-00852-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bianchi RM, et al. 2019. Pathological and microbiological characterization of mastitis in dairy cows. Trop Anim Health Prod. 51(7):2057–2066. 10.1007/s11250-019-01907-0 [DOI] [PubMed] [Google Scholar]
  10. Bonyek-Silva I, et al. 2020. Unbalanced production of LTB(4)/PGE(2) driven by diabetes increases susceptibility to cutaneous leishmaniasis. Emerg Microbes Infect. 9(1):1275–1286. 10.1080/22221751.2020.1773744 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cai J, et al. 2020. Staphylococcus aureus facilitates its survival in bovine macrophages by blocking autophagic flux. J Cell Mol Med. 24(6):3460–3468. 10.1111/jcmm.15027 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Campos B, et al. 2022. Diversity and pathogenesis of Staphylococcus aureus from bovine mastitis: current understanding and future perspectives. BMC Vet Res. 18(1):115. 10.1186/s12917-022-03197-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Caroline V, Sushrut A, Niamh G, Katherine W, Richard OK.. 2009. Mastitis detection: current trends and future perspectives. Trends Biotechnol. 27:486–493. [DOI] [PubMed] [Google Scholar]
  14. Chen F, et al. 2024. VEGF-FGF signaling activates quiescent CD63(+) liver stem cells to proliferate and differentiate. Adv Sci (Weinh). 11(33):e2308711. 10.1002/advs.202308711 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chen H, et al. 2024. Effects of yeast culture on growth performance, immune function, antioxidant capacity and hormonal profile in Mongolian ram lambs. Front Vet Sci. 11:1424073. 10.3389/fvets.2024.1424073 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ciloglu FU, et al. 2021. Drug-resistant Staphylococcus aureus bacteria detection by combining surface-enhanced Raman spectroscopy (SERS) and deep learning techniques. Sci Rep. 11(1):18444. 10.1038/s41598-021-97882-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fu C, et al. 2020. Prostaglandin F(2α)-PTGFR signaling promotes proliferation of endometrial epithelial cells of cattle through cell cycle regulation. Anim Reprod Sci. 213:106276. 10.1016/j.anireprosci.2020.106276 [DOI] [PubMed] [Google Scholar]
  18. Gao Y, et al. 2022. Dual signal light detection of beta-lactoglobulin based on a porous silicon bragg mirror. Biosens Bioelectron. 204:114035. 10.1016/j.bios.2022.114035 [DOI] [PubMed] [Google Scholar]
  19. Geng N, et al. 2020. Autophagy of bovine mammary epithelial cell induced by intracellular Staphylococcus aureus. J Microbiol. 58(4):320–329. 10.1007/s12275-020-9182-8 [DOI] [PubMed] [Google Scholar]
  20. Hind LE, Dembo M, Hammer DA.. 2015. Macrophage motility is driven by frontal-towing with a force magnitude dependent on substrate stiffness. Integr Biol (Camb). 7(4):447–453. 10.1039/c4ib00260a [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hu M, et al. 2017. IL-1β-induced NF-κB activation down-regulates miR-506 expression to promotes osteosarcoma cell growth through JAG1. Biomed Pharmacother. 95:1147–1155. 10.1016/j.biopha.2017.08.120 [DOI] [PubMed] [Google Scholar]
  22. Ilari S, et al. 2020. Protective effect of antioxidants in nitric oxide/COX-2 interaction during inflammatory pain: the role of nitration. Antioxidants (Basel). 9(12):1284. 10.3390/antiox9121284 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Imaizumi N, et al. 2024. Innate immune response of bovine mammary epithelial cells in Mycoplasma bovis mastitis using an in vitro model of bovine mammary gland infection. J Vet Med Sci. 86(7):712–720. 10.1292/jvms.24-0097 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Islam MA, et al. 2020. Transcriptome analysis of the inflammatory responses of bovine mammary epithelial cells: exploring immunomodulatory target genes for bovine mastitis. Pathogens. 9;9(3):200. 10.3390/pathogens9030200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Janjic JM, et al. 2018. Low-dose NSAIDs reduce pain via macrophage targeted nanoemulsion delivery to neuroinflammation of the sciatic nerve in rat. J Neuroimmunol. 318:72–79. 10.1016/j.jneuroim.2018.02.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jimenez-Duran G, Triantafilou M.. 2021. Metabolic regulators of enigmatic inflammasomes in autoimmune diseases and crosstalk with innate immune receptors. Immunology. 163(4):348–362. 10.1111/imm.13326 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kawai T, Akira S.. 2010. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 11(5):373–384. 10.1038/ni.1863 [DOI] [PubMed] [Google Scholar]
  28. Ke Y, Chen Z, Yang R.. 2013. Yersinia pestis: mechanisms of entry into and resistance to the host cell. Front Cell Infect Microbiol. 3:106. 10.3389/fcimb.2013.00106 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kuehl FA, Jr, Egan RW.. 1980. Prostaglandins, arachidonic acid, and inflammation. Science. 210:978–984. [DOI] [PubMed] [Google Scholar]
  30. Li L, Zhao X.. 2018. Characterization of the resistance class 1 integrons in Staphylococcus aureus isolates from milk of lactating dairy cattle in Northwestern China. BMC Vet Res. 14(1):59. 10.1186/s12917-018-1376-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liao Z, et al. 2023. Transcriptomic analyses reveal the potential antibacterial mechanism of citral against Staphylococcus aureus. Front Microbiol. 14:1171339. 10.3389/fmicb.2023.1171339 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Liu B, et al. 2021. Proliferation of bovine endometrial epithelial cells is promoted by prostaglandin E(2)-PTGER2 signaling through cell cycle regulation. Prostaglandins Leukot Essent Fatty Acids. 174:102362. 10.1016/j.plefa.2021.102362 [DOI] [PubMed] [Google Scholar]
  33. Liu K, et al. 2020. Characterization of Staphylococcus aureus isolates from cases of clinical bovine mastitis on large-scale Chinese dairy farms. Front Vet Sci. 7:580129. 10.3389/fvets.2020.580129 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Liu PW, et al. 2025. A pyroptosis-related LncRNA signature for predicting prognosis, immune features and drug sensitivity in ovarian cancer. OTT. 18:585–601. 10.2147/ott.s491130 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Malak HA, et al. 2020. Immune system evasion mechanisms in Staphylococcus aureus: current understanding. J Pure Appl Microbiol. 14(4):2219–2234. 10.22207/JPAM.14.4.01 [DOI] [Google Scholar]
  36. Martinon F, Mayor A, Tschopp J.. 2009. The inflammasomes: guardians of the body. Annu Rev Immunol. 27(1):229–265. 10.1146/annurev.immunol.021908.132715 [DOI] [PubMed] [Google Scholar]
  37. Masters SL, Simon A, Aksentijevich I, Kastner DL.. 2009. Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease. Annu Rev Immunol. 27(1):621–668. 10.1146/annurev.immunol.25.022106.141627 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Matthew SJM, et al. 2018. Targeting the NLRP3 inflammasome in inflammatory diseases. Nat Rev Drug Discov. 17(9):688. 10.1038/nrd.2018.149 [DOI] [PubMed] [Google Scholar]
  39. Mohammad M, et al. 2022. Staphylococcus aureus lipoproteins in infectious diseases. Front Microbiol. 13:1006765. 10.3389/fmicb.2022.1006765 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Muñoz-Planillo R, Franchi L, Miller LS, Núñez G.. 2009. A critical role for hemolysins and bacterial lipoproteins in Staphylococcus aureus-induced activation of the Nlrp3 inflammasome. J Immunol. 183(6):3942–3948. 10.4049/jimmunol.0900729 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Neelam. et al. 2022. Virulence and antimicrobial resistance gene profiles of Staphylococcus aureus associated with clinical mastitis in cattle. PLos One. 17(5):e0264762. 10.1371/journal.pone.0264762 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nguyen M-T, et al. 2017. Lipid moieties on lipoproteins of commensal and non-commensal staphylococci induce differential immune responses. Nat Commun. 8(1):2246. 10.1038/s41467-017-02234-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Gruet P, Maincent P, Berthelot X, Kaltsatos V.. 2001. Bovine mastitis and intramammary drug delivery: review and perspectives. Adv Drug Deliv Rev. 50(3):245–59. 10.1016/S0169-409X(01)00160-0 [DOI] [PubMed] [Google Scholar]
  44. Paerewijck O, Lamkanfi M.. 2022. The human inflammasomes. Mol Aspects Med. 88:101100. 10.1016/j.mam.2022.101100 [DOI] [PubMed] [Google Scholar]
  45. Park JY, Pillinger MH, Abramson SB.. 2006. Prostaglandin E2 synthesis and secretion: the role of PGE2 synthases. Clin Immunol. 119(3):229–240. 10.1016/j.clim.2006.01.016 [DOI] [PubMed] [Google Scholar]
  46. Pidwill GR, Gibson JF, Cole J, Renshaw SA, Foster SJ.. 2020. The role of macrophages in Staphylococcus aureus infection. Front Immunol. 11:620339. 10.3389/fimmu.2020.620339 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ricciotti E, FitzGerald G.. 2011. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol. 31(5):986–1000. 10.1161/ATVBAHA.110.207449 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ruslan M. 2008. Origin and physiological roles of inflammation. Nature. 454(7203):428–35. 10.1038/nature07201 [DOI] [PubMed] [Google Scholar]
  49. Ryman VE, Packiriswamy N, Sordillo LM.. 2015. Role of endothelial cells in bovine mammary gland health and disease. Anim Health Res Rev. 16(2):135–149. 10.1017/S1466252315000158 [DOI] [PubMed] [Google Scholar]
  50. Serhan CN, Levy B.. 2003. Success of prostaglandin E2 in structure-function is a challenge for structure-based therapeutics. Proc Natl Acad Sci USA. 100(15):8609–8611. 10.1073/pnas.1733589100 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shen Y, et al. 2019. Prostaglandin E2 promotes Pam3CSK4-induced inflammation in endometrial epithelial cells of cattle. Anim Reprod Sci. 200:51–59. 10.1016/j.anireprosci.2018.11.010 [DOI] [PubMed] [Google Scholar]
  52. Stenzel W, et al. 2008. Both TLR2 and TLR4 are required for the effective immune response in Staphylococcus aureus-induced experimental murine brain abscess. Am J Pathol. 172(1):132–145. 10.2353/ajpath.2008.070567 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stoll H, Dengjel J, Nerz C, Götz F.. 2005. Staphylococcus aureus deficient in lipidation of prelipoproteins is attenuated in growth and immune activation. Infect Immun. 73(4):2411–2423. 10.1128/IAI.73.4.2411-2423.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sun W, et al. 2017. Selenium suppresses inflammation by inducing microRNA-146a in Staphylococcus aureus-infected mouse mastitis model. Oncotarget. 8(67):110949–110964. 10.18632/oncotarget.20740 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Thomas AW, Ajay C, Jeffrey WP.. 2013. Macrophage biology in development, homeostasis and disease. Nature. 496(7446):445–55. 10.1038/nature12034 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Timothy JF, Joan AG, Vannakambadi KG, Magnus H.. 2014. Adhesion, invasion and evasion: the many functions of the surface proteins of Staphylococcus aureus. Nat Rev Microbiol. 12(1):49–62. 10.1038/nrmicro3161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Trinchieri G, Sher A.. 2007. Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol. 7(3):179–190. 10.1038/nri2038 [DOI] [PubMed] [Google Scholar]
  58. Ullah N, et al. 2021. Genomic investigation of methicillin-resistant Staphylococcus aureus ST113 strains isolated from tertiary care hospitals in Pakistan. Antibiotics. 10(9):1121. 10.3390/antibiotics10091121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Vane JR, Botting RM.. 2003. The mechanism of action of aspirin. Thromb Res. 110(5-6):255–258. 10.1016/s0049-3848(03)00379-7 [DOI] [PubMed] [Google Scholar]
  60. Wang JP, et al. 2021. Differential expression profiles of lncRNA following LPS-induced inflammation in bovine mammary epithelial cells. Front Vet Sci. 8:758488. 10.3389/fvets.2021.758488 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wang X, Eagen WJ, Lee JC.. 2020. Orchestration of human macrophage NLRP3 inflammasome activation by Staphylococcus aureus extracellular vesicles. Proc Natl Acad Sci USA. 117(6):3174–3184. 10.1073/pnas.1915829117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wu F, et al. 2024. Lactococcus garvieae exerts a critical role in inducing inflammation in dairy mastitis by triggering NLRP3 inflammasome-mediated pyroptosis in MAC-T cells. World J Microbiol Biotechnol. 40(4):132. 10.1007/s11274-024-03947-7 [DOI] [PubMed] [Google Scholar]
  63. Wu J, et al. 2020. Prostaglandin E2 regulates activation of mouse peritoneal macrophages by Staphylococcus aureus through Toll-like receptor 2, Toll-like receptor 4, and NLRP3 inflammasome signaling. J Innate Immun. 12(2):154–169. 10.1159/000499604 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yang Y, Lv S, Wang Z, Liu J.. 2022. Selenium ameliorates S. aureus-induced inflammation in bovine mammary epithelial cells by regulating ROS-induced NLRP3 inflammasome. Biol Trace Elem Res. 200(7):3171–3175. 10.1007/s12011-021-02924-7 [DOI] [PubMed] [Google Scholar]
  65. Zhang C, et al. 2022. A microsatellite DNA-derived oligodeoxynucleotide attenuates lipopolysaccharide-induced acute lung injury in mice by inhibiting the HMGB1-TLR4-NF-κB signaling pathway. Front Microbiol. 13:964112. 10.3389/fmicb.2022.964112 [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zhang K, et al. 2023. Staphylococcus aureus increases prostaglandin E(2) secretion in cow neutrophils by activating TLR2, TLR4, and NLRP3 inflammasome signaling pathways. Front Microbiol. 14:1163261. 10.3389/fmicb.2023.1163261 [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Zhang Y, et al. 2025. Dual recombinase polymerase amplification system combined with lateral flow immunoassay for simultaneous detection of Staphylococcus aureus and Vibrio parahaemolyticus. J Pharm Biomed Anal. 255:116621. 10.1016/j.jpba.2024.116621 [DOI] [PubMed] [Google Scholar]
  68. Zhao P, et al. 2024. Biological characterization and genomic analysis of a novel methicillin-resistant Staphylococcus aureus phage, SauPS-28. Microbiol Spectr. 12(2):e0029523. 10.1128/spectrum.00295-23 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1.docx
TVEQ_A_2615759_SM6152.docx (14.9KB, docx)

Data Availability Statement

The data for this study are available by contacting the corresponding authors upon reasonable request.

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