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: Cellular and Molecular Life Sciences: CMLS

Article Title: Cathelicidin-Ka, the first frog-derived TLR2 and TLR4 agonist, induces macrophage activation and promotes inflammation

doi: 10.1007/s00018-025-06068-y

Figure Lengend Snippet: Cath-Ka directly binds to both TLR2 and TLR4. ( A ) Flow cytometry measurement of the binding reaction of FITC-labeled Cath-Ka (1.25–20 µM) with RAW264.7 cells. ( B ) The effects of TLR inhibitors on Cath-Ka-induced TNF-α production. RAW264.7 cells were pre-treated with the indicated inhibitors for 30 min and then co-cultured with Cath-Ka (10 µM) for 24 h before TNF-α production was measured by ELISA. ( C ) The effects of Cath-Ka (10 µM) on LTA (10 µg/mL) - or LPS (100 ng/mL)- induced TNF-α production in RAW264.7 cells. ( D ) Representative SPRi sensograms of Cath-Ka binding to TLR2 or TLR4 immobilized on a gold chip. Recombinant human TLR2 and TLR4 (1 mg/mL) were immobilized on an activated bare gold SPRi chip before Cath-Ka (0.25–4 µM) was used as the mobile phase. ( E ) Representative WB images of TLR2 protein pulled down by Cath-Ka. TLR2 protein of RAW264.7 cell lysates was captured with biotin-tagged Cath-Ka-bound Streptavidin resin before it was eluted and used for WB analyses. Cath-Ka-1 was used as the negative control. ( F ) Representative WB images of CETSA. RAW264.7 cells were stimulated with Cath-Ka (10 µM) or PBS for 1 h under the indicated temperatures before TLR2 (a) and TLR4 (b) protein expressions were detected by WB. Data are expressed as mean ± SD ( n = 3). *** P < 0.001 is thought statistically significant to the control group, while # P < 0.05, ## P < 0.01, and ### P < 0.001 are deemed to have significant differences to the corresponding Cath-Ka/LTA/LPS treatment groups; ns means not significant

Article Snippet: The inhibitors against TLR2 (C29, #HY-100461), TLR3 (CU-CPT 4a, #HY-108473), TLR4 (TAK-242, #HY-11109), TLR5 (TH1020, #HY-116961), TLR7/9 (Hydroxychloroquine, #HY-W031727), TLR7/8 (Enpatoran, #HY-134581), ERK (U0126, # HY-12031 A), p38 (SB203580H, # Y-10256), and JNK (SP600125, #HY-12041) were purchased from MedChem Express (Shanghai, China).

Techniques: Flow Cytometry, Binding Assay, Labeling, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant, Negative Control, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cathelicidin-Ka, the first frog-derived TLR2 and TLR4 agonist, induces macrophage activation and promotes inflammation

doi: 10.1007/s00018-025-06068-y

Figure Lengend Snippet: Cath-Ka activates TLR2/4-MyD88-MAPKs signaling pathways in vitro and vivo. ( A , B ) Representative WB images (left) and quantitative analysis (right) of the expression of TLR2/4-MyD88-MAPKs signaling proteins activated by Cath-Ka. ( A ) RAW264.7 cells were stimulated with Cath-Ka (10 µM) for the indicated time (0.5, 1, 2, 4, and 6 h); ( B ) RAW264.7 cells were stimulated with the indicated concentrations of Cath-Ka (2.5, 5, 10, and 20 µM) for 4 h (for the detection of TLR2, TLR4, and MyD88) or 0.5 h (for the detection of MAPKs) before the proteins related to TLR2/4-MyD88-MAPKs signaling pathways were measured by WB. ( C , D ) Representative WB images (left) and quantitative analysis (right) of the effects of C29 and TAK-242 on the expression of proteins related to TLR2/4-MyD88-MAPKs signaling pathways in the presence of Cath-Ka. RAW264.7 cells were pre-treated with 10 µM C29 ( C ) or 10 µM TAK-242 ( D ) for 30 min and then were cultured with medium containing Cath-Ka (10 µM) for 4 h (for the detection of TLR2, TLR4, and MyD88) or for 0.5 h (for the detection of MAPKs) before the proteins of TLR2/4-MyD88-MAPKs signaling pathways were measured by WB. ( E ) Representative WB images (left) and quantitative analysis (right) of TLR2/4-MyD88-MAPKs signaling protein expression in peritoneal cells activated by Cath-Ka. Data are expressed as mean ± SD ( n = 3–4). * P < 0.05, ** P < 0.01, and *** P < 0.001 are thought statistically significant to the control group, while # P < 0.05, ## P < 0.01, and ### P < 0.001 are deemed to have significant differences between the corresponding Cath-Ka treatment groups; ns means not significant

Article Snippet: The inhibitors against TLR2 (C29, #HY-100461), TLR3 (CU-CPT 4a, #HY-108473), TLR4 (TAK-242, #HY-11109), TLR5 (TH1020, #HY-116961), TLR7/9 (Hydroxychloroquine, #HY-W031727), TLR7/8 (Enpatoran, #HY-134581), ERK (U0126, # HY-12031 A), p38 (SB203580H, # Y-10256), and JNK (SP600125, #HY-12041) were purchased from MedChem Express (Shanghai, China).

Techniques: Protein-Protein interactions, In Vitro, Expressing, Cell Culture, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Cathelicidin-Ka, the first frog-derived TLR2 and TLR4 agonist, induces macrophage activation and promotes inflammation

doi: 10.1007/s00018-025-06068-y

Figure Lengend Snippet: Identification of the structure and function relation of Cath-Ka. ( A ) The interaction mode of Cath-Ka and TLR4 predicted by AlphaFold2 server and visualized by Pymol. ( B ) Hydrophobic interactions and hydrogen bond (green dashed line) of Cath-Ka-TLR4 complex around four residues at the N-terminus of Cath-Ka. ( C ) Comparison of their primary sequences and physicochemical parameters. The two cysteine of Cath-Ka-1 does not form the disulfide bond. ( D ) Secondary structure of Cath-Ka and its analogues in SDS solution. ( E - G ) Effect comparison of 10 µM Cath-Ka and its analogues on macrophage morphology ( E ), proliferation ( F ) and TNF-α production ( G ). Scale bar = 50 μm in Figure G. Data are expressed as mean ± SD ( n = 3). * : P < 0.05, ** : P < 0.01, and *** : P < 0.001 vs. Control; # : P < 0.05, ## : P < 0.01, and ### : P < 0.001 vs. Cath-Ka treatment; ns: Not significant

Article Snippet: The inhibitors against TLR2 (C29, #HY-100461), TLR3 (CU-CPT 4a, #HY-108473), TLR4 (TAK-242, #HY-11109), TLR5 (TH1020, #HY-116961), TLR7/9 (Hydroxychloroquine, #HY-W031727), TLR7/8 (Enpatoran, #HY-134581), ERK (U0126, # HY-12031 A), p38 (SB203580H, # Y-10256), and JNK (SP600125, #HY-12041) were purchased from MedChem Express (Shanghai, China).

Techniques: Comparison, Analogues, Control

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