lps isolated from e. coli 0111:b4  (Millipore)

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Priming from within: TLR2 dependent but receptor independent activation of the mammary macrophage inflammasome by Streptococcus uberis

doi: 10.3389/fcimb.2024.1444178

Figure Lengend Snippet: S. uberis SUB1154 protein is involved in the production of IL-1β from BMMOs. Bovine mammary macrophages (BMMOs) were isolated from milk and seeded into culture dishes at 50,000 BMMOs/well. BMMOs were challenged with either heat-killed S. uberis strain 0140J or SUB1154 deletion mutant (0140JΔ sub1154 ) at a multiplicity of infection (MOI) of 50:1 bacterium:BMMO and/or 2 nM rSUB1154 or rSUB1154NP (proteolytically compromised) protein. Supernatants were collected 20h after challenge and the concentration of IL-1β was measured by ELISA. BMMOs were unstimulated in the no treatment (NT) group and this mean was deducted from the other values, which were then standardised to the LPS (10 ng/mL) positive control. Data is presented as N=3 ± SD. Data was statistically analysed using a one-way ANOVA followed by Tukey multiple comparisons post hoc test (**** P< 0.0001).

Article Snippet: We previously found ( ) that heat-killing S. uberis did not significantly perturb the macrophage response, but enabled us to have fine control over the multiplicity of infection in our model. Heat-killed S. uberis strains at a multiplicity of infection (MOI) of 50:1 bacterium:BMMO; 10 ng/mL LPS (isolated from E. coli 0111:B4, Millipore, LPS25); 2 nM rSUB1154/NP; inflammasome primer 1.0 μg/mL Pam3CSK4 (Tocris, 4633); inflammasome activator 500 μg/mL silica (Sigma, 421553); cell entry inhibitor 10 μM Cytochalasin D (CyD) (Tocris, 1233) for 2h prior to additional stimuli for 20h; TLR2 inhibitor 100 μM C29 (Adooq Bioscience, A17160) for 1h prior to additional stimuli for 20h.

Techniques: Isolation, Mutagenesis, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Priming from within: TLR2 dependent but receptor independent activation of the mammary macrophage inflammasome by Streptococcus uberis

doi: 10.3389/fcimb.2024.1444178

Figure Lengend Snippet: S. uberis SUB1154 protein functions intracellularly to prime the NLRP3 inflammasome in BMMOs. Bovine mammary macrophages (BMMOs) were isolated from milk and seeded into culture dishes at 50,000 BMMOs/well. BMMOs were challenged with either heat-killed S. uberis strain 0140J or SUB1154 deletion mutant (0140JΔ sub1154 ) at a multiplicity of infection (MOI) of 50:1 bacterium:BMMO and/or 2 nM rSUB1154 protein. Cell entry was inhibited by incubating BMMOs with 10 µM Cytochalasin D (CyD) for 2h. Supernatants were collected 20h after challenge and the concentration of IL-1β was measured by ELISA. BMMOs were unstimulated in a no treatment group and the mean of this group was deducted from the other values and standardised to the LPS positive control (10 ng/mL). Data is presented as N=9 ± SD. Data was statistically analysed using a one-way ANOVA followed by Tukey multiple comparisons post hoc test ****=p<0.0001.

Article Snippet: We previously found ( ) that heat-killing S. uberis did not significantly perturb the macrophage response, but enabled us to have fine control over the multiplicity of infection in our model. Heat-killed S. uberis strains at a multiplicity of infection (MOI) of 50:1 bacterium:BMMO; 10 ng/mL LPS (isolated from E. coli 0111:B4, Millipore, LPS25); 2 nM rSUB1154/NP; inflammasome primer 1.0 μg/mL Pam3CSK4 (Tocris, 4633); inflammasome activator 500 μg/mL silica (Sigma, 421553); cell entry inhibitor 10 μM Cytochalasin D (CyD) (Tocris, 1233) for 2h prior to additional stimuli for 20h; TLR2 inhibitor 100 μM C29 (Adooq Bioscience, A17160) for 1h prior to additional stimuli for 20h.

Techniques: Isolation, Mutagenesis, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Priming from within: TLR2 dependent but receptor independent activation of the mammary macrophage inflammasome by Streptococcus uberis

doi: 10.3389/fcimb.2024.1444178

Figure Lengend Snippet: SUB1154 primes the inflammasome by interacting with intracellular TIR domains. Bovine mammary macrophages (BMMOs) were isolated from milk and seeded into culture dishes at 50,000 BMMOs/well. BMMOs were incubated in the presence and absence of the TLR2 inhibitors C29 (intracellularly binds to the TIR (toll-interleukin receptor) domain; 100 µM) or MMG 11 (antagonist to extracellular binding of TLR2; 100 µM) 1h prior to subsequent challenge with either heat-killed S. uberis strain 0140J or SUB1154 deletion mutant (0140JΔ sub1154 ) at a multiplicity of infection (MOI) of 50:1 bacterium:BMMO and/or 2 nM rSUB1154 or rSUB1154NP (proteolytically compromised) protein; 1.0 µg/mL Pam3CSK4 (primes the inflammasome) and/or 500 µg/mL silica (activates the inflammasome). IL-1β concentration was measured from the supernatants after 20h by ELISA and values were standardised to LPS. Data is presented as N=3 ± SD, statistically analysed using a one-way ANOVA followed by Tukey multiple comparisons post hoc test ****=p<0.0001, ns, not significant, comparisons against 0140J alone. ND, none detected.

Article Snippet: We previously found ( ) that heat-killing S. uberis did not significantly perturb the macrophage response, but enabled us to have fine control over the multiplicity of infection in our model. Heat-killed S. uberis strains at a multiplicity of infection (MOI) of 50:1 bacterium:BMMO; 10 ng/mL LPS (isolated from E. coli 0111:B4, Millipore, LPS25); 2 nM rSUB1154/NP; inflammasome primer 1.0 μg/mL Pam3CSK4 (Tocris, 4633); inflammasome activator 500 μg/mL silica (Sigma, 421553); cell entry inhibitor 10 μM Cytochalasin D (CyD) (Tocris, 1233) for 2h prior to additional stimuli for 20h; TLR2 inhibitor 100 μM C29 (Adooq Bioscience, A17160) for 1h prior to additional stimuli for 20h.

Techniques: Isolation, Incubation, Binding Assay, Mutagenesis, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Priming from within: TLR2 dependent but receptor independent activation of the mammary macrophage inflammasome by Streptococcus uberis

doi: 10.3389/fcimb.2024.1444178

Figure Lengend Snippet: S. uberis SUB1154 protein primes the NLRP3 inflammasome in BMMOs. Bovine mammary macrophages (BMMOs) at 50,000 BMMOs/well were challenged with heat-killed S. uberis strain 0140J or SUB1154 deletion mutant (0140JΔ sub1154 ) at a multiplicity of infection (MOI) of 50:1 bacterium:BMMO and/or 2 nM rSUB1154 or rSUB1154NP (mutant predicted protease site) protein. Supernatants were collected 20h after challenge and the concentration of IL-1β measured by ELISA. BMMOs were unstimulated in a no treatment group and the mean of this group was deducted from the other values and standardised to the LPS positive control (10 ng/mL). Data is presented as N=3 ± SD. In addition to S. uberis and rSUB1154 and NP proteins, BMMOs were challenged with 1.0 µg/mL Pam3CSK4 (primes the inflammasome) and 500 µg/mL silica (activates the inflammasome). Data was statistically analysed using a one-way ANOVA followed by Tukey multiple comparisons post hoc test (** P< 0.01 compared to silica + rSUB1154).

Article Snippet: We previously found ( ) that heat-killing S. uberis did not significantly perturb the macrophage response, but enabled us to have fine control over the multiplicity of infection in our model. Heat-killed S. uberis strains at a multiplicity of infection (MOI) of 50:1 bacterium:BMMO; 10 ng/mL LPS (isolated from E. coli 0111:B4, Millipore, LPS25); 2 nM rSUB1154/NP; inflammasome primer 1.0 μg/mL Pam3CSK4 (Tocris, 4633); inflammasome activator 500 μg/mL silica (Sigma, 421553); cell entry inhibitor 10 μM Cytochalasin D (CyD) (Tocris, 1233) for 2h prior to additional stimuli for 20h; TLR2 inhibitor 100 μM C29 (Adooq Bioscience, A17160) for 1h prior to additional stimuli for 20h.

Techniques: Mutagenesis, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Priming from within: TLR2 dependent but receptor independent activation of the mammary macrophage inflammasome by Streptococcus uberis

doi: 10.3389/fcimb.2024.1444178

Figure Lengend Snippet: BMMO differential RNA expression of inflammasome pathway genes in response to S. uberis stimulation. RNA was extracted from isolated bovine mammary macrophages (BMMOs) at 0, 2, 4, 8, 12, 16 and 20h after challenge with either 10 ng/mL LPS; heat-killed S. uberis strain 0140J or SUB1154 deletion mutant (0140JΔ sub1154 ) at a multiplicity of infection (MOI) of 50:1 bacterium:BMMO; 2 nM rSUB1154 or rSUB1154NP (proteolytically compromised) protein. Changes in mRNA quantity were determined by real time reverse transcription quantitative PCR using 3 reference genes (GAPDH, ACTB and RPL13a) for 4 target genes: TLR2 (A) , NF-kB (B) , pro-caspase-1 (C) and pro-IL-1β (D) . Log 2 was calculated and the data is presented as N=3 ± SD. Values at 0h were used to determine baseline mRNA levels (0 Log 2 ). Asterisks denote significance calculated with Two-Way ANOVA and Dunnett’s multiple comparisons test, compared to 0140J, at 4h and 20h; * P< 0.05, ** P< 0.01,*** P< 0.001 **** P< 0.0001. All timepoint statistics available in Supplementary Table 1 .

Article Snippet: We previously found ( ) that heat-killing S. uberis did not significantly perturb the macrophage response, but enabled us to have fine control over the multiplicity of infection in our model. Heat-killed S. uberis strains at a multiplicity of infection (MOI) of 50:1 bacterium:BMMO; 10 ng/mL LPS (isolated from E. coli 0111:B4, Millipore, LPS25); 2 nM rSUB1154/NP; inflammasome primer 1.0 μg/mL Pam3CSK4 (Tocris, 4633); inflammasome activator 500 μg/mL silica (Sigma, 421553); cell entry inhibitor 10 μM Cytochalasin D (CyD) (Tocris, 1233) for 2h prior to additional stimuli for 20h; TLR2 inhibitor 100 μM C29 (Adooq Bioscience, A17160) for 1h prior to additional stimuli for 20h.

Techniques: RNA Expression, Isolation, Mutagenesis, Infection, Reverse Transcription, Real-time Polymerase Chain Reaction