Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: 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 (Δ lgt ), or its complemented strain, lgt ::ermB + pRB lgt (+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.

Article Snippet: The bBMMs (5 × 10 6 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 PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Infection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: 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.

Article Snippet: The bBMMs (5 × 10 6 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 PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Infection, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: Host perspective: TLR2, TLR4, and NLRP3 play essential roles in PGE 2 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 PGE 2 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.

Article Snippet: The bBMMs (5 × 10 6 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 PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Infection, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: Cross-talk: PGE 2 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 PGE 2 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.

Article Snippet: The bBMMs (5 × 10 6 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 PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Microscopy

Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: Cross-talk: Excess PGE 2 exacerbates inflammation and impairs intracellular killing in S. aureus -infected bBMMs. bBMMs were pretreated with the PGE 2 (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.

Article Snippet: The bBMMs (5 × 10 6 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 PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Infection, Expressing, Quantitative RT-PCR, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Microscopy

Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: Graphical abstract of the present study: the involvement of TLR2-, TLR4-, and NLRP3-dependent PGE 2 signaling in macrophage responses to S. aureus , which modulates inflammatory signaling and phagocytic activity and thereby contributes to the pathogenesis of bovine mastitis.

Article Snippet: The bBMMs (5 × 10 6 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 PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Activity Assay

Journal: Life

Article Title: Exploratory Analysis of TLR2, TLR4, Interleukin 6 and Interleukin 10 Gene Polymorphisms in Relation to Clinical Early-Onset Sepsis in Preterm Neonates: A Single-Center Study

doi: 10.3390/life16010103

Figure Lengend Snippet: ( a ) TLR2 - Arg753Gln , TLR4 - Asp299Gly , IL6 - 174G/C , and IL10 - 1082G/A polymorphisms identification. TLR2 - Arg753Gln polymorphism: lane 1—pBRHaeIIIDigest DNA molecular marker; lanes 2, 6, and 8—Gln/Gln genotype; lanes 3, 4, 7, and 10—Arg/Arg genotype; and lanes 5 and 9—Arg/Gln genotype. ( b ) TLR4 - Asp299Gly polymorphism: lane 1—pBRHAeIII Digest DNA molecular marker, lanes 2, 6, and 10—Asp/Asp genotype, lanes 3, 4, 5, 7, and 9—Asp/Gly genotype, and lane 8—Gly/Gly genotype; ( c ) IL6 - 174G/C polymorphism: lane 1—pBRHaeIIIDigest DNA molecular marker, lanes 2 and 6—GG genotype, lanes 3, 5, 8, 9, and 10—GC genotype, and lanes 4 and 7—CC genotype. ( d ) IL10 -1082G/A polymorphism: lane 1—pBRHaeIIIDigest DNA molecular marker, lane 2—PCR fragment, lane 3—AA genotype, lanes 4, 5, 7, 8, and 10—GA genotype, and lanes 6 and 9—GG genotype.

Article Snippet: An amount of 6 μL of amplified DNA was digested with 2 units of MspI (TLR2) , NcoI (TLR4) , LweI (IL6) , XagI (IL10) , and restriction enzymes (New England Biolabs UK, Ltd., Hitchin, UK).

Techniques: Marker, Ubiquitin Proteomics