tlr2 inhibitor c29  (Miltenyi Biotec)

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: Scientific Reports

Article Title: Phage-mediated TLR2 signaling attenuates intracellular Mycobacterium abscessus survival in macrophages

doi: 10.1038/s41598-025-07320-y

Figure Lengend Snippet: Impact of TLR2/TLR4 signaling activation on MAB infection. ( A ) The intracellular growth dynamics of MAB in TLR2 and TLR4-stimulated THP-1 cells before and after bacterial infection. The bacterial survival rates were assessed through viable CFU counts at 1 h, 24 h, and 72 h after MAB infection. The data is presented as the mean ± SD of three independent experiments, each conducted in triplicate. * p < 0.05, ** p < 0.01 denote statistical significance between the TLR-treated groups and the MAB infection alone group at the corresponding time points. Following the 2 h MAB infection of THP-1 monolayers, either ( B ) TLR2/4 inhibitor oxPAPC, ( C ) TLR2 inhibitor TL2-C29 or ( D ) TLR4 inhibitor CLI-095 from InvivoGen was added to THP-1 cell monolayers for 30 min and then exposed to phages at 10:1 ratio. Intracellular MAB growth was evaluated at day 3 post-infection where the control group did not receive antagonist treatment. * p < 0.05 and ** p < 0.01, denote statistical significance between the TLR-treated and non-treated corresponding groups. The data is presented as the mean ± SD of three independent experiments each performed in three technical replicates.

Article Snippet: Following the 2 h MAB infection of THP-1 monolayers, either ( B ) TLR2/4 inhibitor oxPAPC, ( C ) TLR2 inhibitor TL2-C29 or ( D ) TLR4 inhibitor CLI-095 from InvivoGen was added to THP-1 cell monolayers for 30 min and then exposed to phages at 10:1 ratio.

Techniques: Activation Assay, Infection, Control

Journal: Scientific Reports

Article Title: Phage-mediated TLR2 signaling attenuates intracellular Mycobacterium abscessus survival in macrophages

doi: 10.1038/s41598-025-07320-y

Figure Lengend Snippet: Evaluation of intracellular growth of MAB morphotypes in THP-1 cells. ( A ) Macrophages were infected with either MAB-S or MAB-R at 1:1 MOI for 2 h, washed three times and then either left untreated (control group) or exposed with TLR2 agonist or treated with Ph17 for 1 h (experimental groups). After treatment, cell monolayers were gently washed three times and replenished with fresh RPMI 1640 media. THP-1 cell were lysed in 0.1% of TritonX-100 and via mechanical scraping at 1 h, 24 h and 72 h post-infection, and each well were serially diluted and plated on 7H10 agar plates for CFU quantification. ** p < 0.01, denote statistical significance between MAB-S only infection and MAB-S/TLR2 or MAB-S/Ph17 treated groups at day 3 as well as between MAB-R only infection and MAB-R/Ph17 treated groups at 24 h and 72 h post-infection. The data is presented as the mean ± SD of three experiments in four technical replicates. ( B ) THP-1 cell monolayers were infected with either MAB-S or MAB-R at 1:1 MOI for 2 h, washed three times and either incubated with TLR2 agonist or treated with Ph17 for 1 h. After treatment, cell monolayers were gently washed three times and replenished with fresh RPMI 1640 media. At day 3 post-infection, supernatants were removed and processed separately from the cell lysates for CFU counts. Each sample (supernatant as well as cell lysate) were serially diluted and plated on 7H10 agar plates. Bacterial colonies were counted after 5 days of incubation at 37 °C. ** p < 0.01, denote statistical significance between extracellular and intracellular MAB counts within the same experimental group. The data is presented as the mean ± SD of three experiments in three technical replicates. ( C ) Ultrathin sections of THP-1 human macrophages were obtained after infection with MAB-S or ( D ) MAB-R at 1:1 MOI for 2 h and then treating with Ph17 at 10:1 ration for 1 h. Three hours later, cells were washed, fixed in the Karnovsky fixative, and processed at the Oregon State University Electron Microscopy facility. TEM studies were conducted three times, with four representative images provided for each MAB morphotypes are presented. Vacuole merging sites are indicated by red arrows. B for Bacteria and Ph-Phage.

Article Snippet: Following the 2 h MAB infection of THP-1 monolayers, either ( B ) TLR2/4 inhibitor oxPAPC, ( C ) TLR2 inhibitor TL2-C29 or ( D ) TLR4 inhibitor CLI-095 from InvivoGen was added to THP-1 cell monolayers for 30 min and then exposed to phages at 10:1 ratio.

Techniques: Infection, Control, Incubation, Electron Microscopy, Bacteria

Journal: Scientific Reports

Article Title: Phage-mediated TLR2 signaling attenuates intracellular Mycobacterium abscessus survival in macrophages

doi: 10.1038/s41598-025-07320-y

Figure Lengend Snippet: The NLRP3 inflammasome and ROS activation in MAB-S infected and Ph17 treated macrophages. Gene expression of ( A ) NLRP3, ( B ) NF-kB, and ( C ) GILZ were measured by qRT-PCR at 24 h post-infection with MAB-S. TLR2 treated group served as a positive control and MAB-S only infection as a negative control. The relative fold change in gene expression is calculated using uninfected THP-1 cells as the control. ( D ) ROS levels were measured over 24 h of bacterial infection of THP-1 cells for experimental and control groups including uninfected cells using CellROX Green at 485/520 nm according to manufacturer’s protocol. The fluorescent intensity in all tested groups were normalized to the uninfected background control. Results are shown for 5 h time point where most changes in ROS are detected. The data are normalized with ROS levels of uninfected/untreated THP-1 cells. ** p < 0.01, denote statistical significance for ( A – D ) between the MAB-infected group and MAB/TLR2 and MAB/Ph17 treated groups. The data is presented as the mean ± SD of three independent experiments each performed in three technical replicates.

Article Snippet: Following the 2 h MAB infection of THP-1 monolayers, either ( B ) TLR2/4 inhibitor oxPAPC, ( C ) TLR2 inhibitor TL2-C29 or ( D ) TLR4 inhibitor CLI-095 from InvivoGen was added to THP-1 cell monolayers for 30 min and then exposed to phages at 10:1 ratio.

Techniques: Activation Assay, Infection, Gene Expression, Quantitative RT-PCR, Positive Control, Negative Control, Control

Journal: Scientific Reports

Article Title: Phage-mediated TLR2 signaling attenuates intracellular Mycobacterium abscessus survival in macrophages

doi: 10.1038/s41598-025-07320-y

Figure Lengend Snippet: The NLRP3 inflammasome activation leads to elevated production of IL-1β and caspase 1 activation in MAB-S infected and Ph17 treated THP-1 cells. ( A ) Supernatants were obtained from macrophages infected with either live or heat-killed MAB-S or treated with Ph17 without bacterial infection at 5 h and 24 h time point. The supernatant obtained from THP-1 without infection or treatment served as a baseline control for the corresponding timepoints. We also collected supernatants from MAB-S infected/TLR2 exposed and MAB-S/Ph17 treated cells and measured IL-1β secretion levels with ELISA. The data are normalized with IL-1β secretion levels of uninfected/untreated THP-1 cells. * p < 0.05 and ** p < 0.01 denote statistical significance between the MAB-infected group and both the MAB/TLR2 and MAB/Ph17 treated groups at the corresponding time points. The data is presented as the mean ± SD of three independent experiments each performed in four technical replicates. ( B ) Cell lysates were obtained from the same experimental and control groups at 24 h and precleared total protein samples were subjected for Western Blot analysis for caspase-1 (pro form at 45 kDa; active form at 10 kDa). The experiments were performed three times, and immunoblot images were recorded on the Odyssey Imager (Li-Cor).

Article Snippet: Following the 2 h MAB infection of THP-1 monolayers, either ( B ) TLR2/4 inhibitor oxPAPC, ( C ) TLR2 inhibitor TL2-C29 or ( D ) TLR4 inhibitor CLI-095 from InvivoGen was added to THP-1 cell monolayers for 30 min and then exposed to phages at 10:1 ratio.

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