Journal: International Journal of Molecular Sciences

Article Title: AhR-Induced Anti-Inflammatory Effects on a Caco-2/THP-1 Co-Culture Model of Intestinal Inflammation Are Mediated by PPARγ

doi: 10.3390/ijms252313072

Figure Lengend Snippet: Effects of inhibitors on FICZ-mediated nuclear translocation and protein expression of AhR in THP-1 cells. Macrophage-differentiated THP-1 cells were treated with 10 µM FICZ, 10 µM GW9662 (GW), and 10 µM CH223191 (CH), either alone or combined, for 24 h. Cells were harvested, fixed, and stained after treatments. Nuclear translocation ( A ) and overall expression ( B ) of AhR were assessed via imaging flow cytometry; representative images of the treatment groups are also shown ( C ). Data are displayed as normalized % of cells of high similarity indexes for translocation and as normalized MFI values for expression. Results are shown as mean ± standard deviation of the mean ( n = 4). Statistical significance was verified by one-way ANOVA followed by Tukey’s post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 in comparison to the untreated control group (first dataset) or LPS-treated group (second and third datasets). “+” indicates the respective treatment at the left side of the graph was carried out; “-” indicates it was not. Dotted vertical lines separate datasets. The small horizontal line groups columns together.

Article Snippet: Once the Caco-2/THP-1 Transwell ® co-culture system was established, in order to evoke agonistic/antagonistic responses, cells were treated with different combinations of pioglitazone (PPARγ-specific agonist) (Sigma Aldrich), FICZ (potent AhR agonist) (MeCDhemExpress, Monmouth Junction, NJ, USA), GW9662 (specific PPARγ antagonist) (Abcam, Cambridge, UK), and CH223191 (specific AhR antagonist) (Tocris, Bristol, UK), all at a final concentration of 10 µM; inhibitors GW9662 and CH223191 were also used at concentrations ranging from 0.5 µM to 10 µM in one specific experiment.

Techniques: Translocation Assay, Expressing, Staining, Imaging, Flow Cytometry, Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: AhR-Induced Anti-Inflammatory Effects on a Caco-2/THP-1 Co-Culture Model of Intestinal Inflammation Are Mediated by PPARγ

doi: 10.3390/ijms252313072

Figure Lengend Snippet: Expression of inflammatory surface markers in THP-1 cells. Macrophage-differentiated THP-1 cells were treated with 100 ng/mL LPS alongside 10 µM pioglitazone (Pio), 10 µM FICZ, 10 µM GW9662, and 10 µM CH223191, either alone or combined, for 24 h. Cells were harvested and stained after treatments. Expression of surface markers TLR4 ( A ), CD80 ( B ), and CD86 ( C ) were assessed by conventional flow cytometry. Data are displayed as normalized MFI values. Results are shown as mean ± standard deviation of the mean ( n = 4). Statistical significance was verified by one-way ANOVA followed by Tukey’s post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 in comparison to the untreated control group (first dataset) or the LPS-treated group (second and third datasets). # p ≤ 0.05 in comparison to the LPS-treated group (first dataset) or the Pio/FICZ-treated group (second and third datasets). “+” indicates the respective treatment at the left side of the graph was carried out; “-” indicates it was not. Dotted vertical lines separate datasets. Small horizontal lines group columns together.

Article Snippet: Once the Caco-2/THP-1 Transwell ® co-culture system was established, in order to evoke agonistic/antagonistic responses, cells were treated with different combinations of pioglitazone (PPARγ-specific agonist) (Sigma Aldrich), FICZ (potent AhR agonist) (MeCDhemExpress, Monmouth Junction, NJ, USA), GW9662 (specific PPARγ antagonist) (Abcam, Cambridge, UK), and CH223191 (specific AhR antagonist) (Tocris, Bristol, UK), all at a final concentration of 10 µM; inhibitors GW9662 and CH223191 were also used at concentrations ranging from 0.5 µM to 10 µM in one specific experiment.

Techniques: Expressing, Staining, Flow Cytometry, Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: AhR-Induced Anti-Inflammatory Effects on a Caco-2/THP-1 Co-Culture Model of Intestinal Inflammation Are Mediated by PPARγ

doi: 10.3390/ijms252313072

Figure Lengend Snippet: Secretion of proinflammatory cytokines by THP-1 cells. Macrophage-differentiated THP-1 cells were treated with 100 ng/mL LPS alongside 10 µM pioglitazone (Pio), 10 µM FICZ, 10 µM GW9662, and 10 µM CH223191, either alone or combined, for 24 h. Supernatants were collected after treatments. Secretion of cytokines TNF-α ( A ), IL-6 ( B ), and IL-10 ( C ) were assessed by ELISA. Data are displayed as [µg/mL] for TNF-α and [pg/mL] for IL-6 and IL-10. Results are shown as mean ± standard deviation of the mean ( n = 4). Statistical significance was verified by one-way ANOVA followed by Tukey’s post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 in comparison to the untreated control group (first dataset) or the LPS-treated group (second and third datasets). # p ≤ 0.05, ## p ≤ 0.01, #### p ≤ 0.0001 in comparison to the LPS-treated group (first dataset) or the Pio/FICZ-treated group (second and third datasets). “+” indicates the respective treatment at the left side of the graph was carried out; “-” indicates it was not. Dotted vertical lines separate datasets. Small horizontal lines group columns together.

Article Snippet: Once the Caco-2/THP-1 Transwell ® co-culture system was established, in order to evoke agonistic/antagonistic responses, cells were treated with different combinations of pioglitazone (PPARγ-specific agonist) (Sigma Aldrich), FICZ (potent AhR agonist) (MeCDhemExpress, Monmouth Junction, NJ, USA), GW9662 (specific PPARγ antagonist) (Abcam, Cambridge, UK), and CH223191 (specific AhR antagonist) (Tocris, Bristol, UK), all at a final concentration of 10 µM; inhibitors GW9662 and CH223191 were also used at concentrations ranging from 0.5 µM to 10 µM in one specific experiment.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: AhR-Induced Anti-Inflammatory Effects on a Caco-2/THP-1 Co-Culture Model of Intestinal Inflammation Are Mediated by PPARγ

doi: 10.3390/ijms252313072

Figure Lengend Snippet: Epithelial integrity and expression of tight junction proteins in Caco-2 cells. Caco-2 cells were placed in a co-culture system alongside macrophage-differentiated THP-1 cells and were treated with 100 ng/mL LPS alongside 10 µM pioglitazone (Pio), 10 µM FICZ, 10 µM GW9662, and 10 µM CH223191, either alone or combined, for 24 h. TEER measurements on the Caco-2 monolayer were performed immediately before and after the treatment period ( A ). Caco-2 cells were also harvested in RIPA buffer, and the expression of the tight junction proteins occludin ( B ), claudin-4 ( C ), and claudin-2 ( D ) was assessed by Western blotting. For TEER measurements, data are displayed as log 2 -fold change, and for blots, as normalized arbitrary units measuring the expression ratio of the target protein and housekeeping protein β-actin. Results are shown as mean ± standard deviation of the mean ( n = 6). Statistical significance was verified by one-way ANOVA followed by Tukey’s post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 in comparison to the untreated control group (first dataset) or the LPS-treated group (second and third datasets). # p ≤ 0.05, ## p ≤ 0.01 in comparison to the LPS-treated group (first dataset) or the Pio/FICZ-treated group (second and third datasets). “+” indicates the respective treatment at the left side of the graph was carried out; “-” indicates it was not. Dotted vertical lines separate datasets. The small horizontal line groups columns together. Lanes within a single blot were rearranged in order to align with the display of experimental groups; thin dotted vertical lines between bands indicate lanes that were spliced together. Original uncropped blots are shown in the . kDa = kilodaltons.

Article Snippet: Once the Caco-2/THP-1 Transwell ® co-culture system was established, in order to evoke agonistic/antagonistic responses, cells were treated with different combinations of pioglitazone (PPARγ-specific agonist) (Sigma Aldrich), FICZ (potent AhR agonist) (MeCDhemExpress, Monmouth Junction, NJ, USA), GW9662 (specific PPARγ antagonist) (Abcam, Cambridge, UK), and CH223191 (specific AhR antagonist) (Tocris, Bristol, UK), all at a final concentration of 10 µM; inhibitors GW9662 and CH223191 were also used at concentrations ranging from 0.5 µM to 10 µM in one specific experiment.

Techniques: Expressing, Co-Culture Assay, Western Blot, Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: AhR-Induced Anti-Inflammatory Effects on a Caco-2/THP-1 Co-Culture Model of Intestinal Inflammation Are Mediated by PPARγ

doi: 10.3390/ijms252313072

Figure Lengend Snippet: Schematics summarizing the interplay between AhR, PPARγ, and ligands in the “leaky gut” Transwell ® co-culture model. Under LPS-elicited inflammatory conditions, TLR4 activation in macrophage-differentiated THP-1 cells ( 1 ) leads to a series of downstream effects, namely, increased secretion of cytokines, such as TNF-α, IL-6, and IL-10 ( 2 ). These cytokines, especially IL-6, once released in the intercellular milieu, come in contact with the Caco-2 epithelial monolayer and disrupt its integrity by upregulating claudin-2 expression ( 3 ). In the presence of the PPARγ-agonist pioglitazone (Pio), all of these effects are attenuated with the exception of increased claudin-2 expression, which nonetheless culminate in reduced inflammatory status in THP-1 macrophages and preserved integrity of the Caco-2 monolayer ( 4 ) (left dashed squares). The AhR-agonist FICZ elicits similar effects while also restoring claudin-2 to basal levels ( 5 ) (right dashed squares) ( A ). In the presence of inhibitors, the anti-inflammatory response elicited by the agonists is mostly hampered, but in different ways ( 6 ). Blocking AhR with CH223191 (CH) prevents the effect of pioglitazone in attenuating IL-6 secretion by THP-1 macrophages, but as pioglitazone still elicits other PPARγ-mediated effects that do not rely on AhR, monolayer permeability is still restored, albeit not necessarily relying on claudin-2 ( 7 ) (left dashed squares). On the other hand, blocking PPARγ with GW9662 (GW) strongly inhibits the actions of FICZ, as it prevents both FICZ-mediated AhR nuclear translocation and also FICZ-mediated effects that do not rely on AhR translocation, which may or may not be AhR-mediated. This leads to increased secretion of IL-6 (and likely of other inflammatory mediators), culminating in increased LPS-mediated claudin-2 expression and subsequent increase in Caco-2 monolayer permeability ( 8 ) (right dashed squares) ( B ).

Article Snippet: Once the Caco-2/THP-1 Transwell ® co-culture system was established, in order to evoke agonistic/antagonistic responses, cells were treated with different combinations of pioglitazone (PPARγ-specific agonist) (Sigma Aldrich), FICZ (potent AhR agonist) (MeCDhemExpress, Monmouth Junction, NJ, USA), GW9662 (specific PPARγ antagonist) (Abcam, Cambridge, UK), and CH223191 (specific AhR antagonist) (Tocris, Bristol, UK), all at a final concentration of 10 µM; inhibitors GW9662 and CH223191 were also used at concentrations ranging from 0.5 µM to 10 µM in one specific experiment.

Techniques: Co-Culture Assay, Activation Assay, Expressing, Blocking Assay, Permeability, Translocation Assay