Fig 1: Blocking RILP cleavage in producer cells imparts a protective effect in target cells. (A) The localization of the RILP-binding protein Rab7 is a surrogate for RILP cleavage. Producer THP-1 cells were treated with 100 ng/ml LPS for 24 h, fixed with 4% PFA and immunostained for Rab7. LPS treatment results in a redistribution of Rab7 throughout the cell periphery, indicating RILP cleavage. This cleavage is confirmed by incubating LPS-treated cells with the caspase-1 inhibitor Z-YVAD-FMK (100 µM). Scale bars: 15 µm. Ut, untreated. (B) ImageJ analysis quantifying the cellular distribution of Rab7 from the nucleus. n=a minimum of 30 cells from three individual experiments. (C) Western blot analysis of THP-1 producer cells that were transduced with lentivirus expressing control mCherry (empty vector) or virus expressing ncRILP–Flag. The dotted line represents a cut in the blot. Image representative of three repeats. (D) When co-cultured with LPS treated macrophages, IL-1ß and TNFa increase dramatically in target THP-1 cells. Expression of ncRILP in treated macrophage completely blocks this response. n=3. (E,F) Similar results occurred in hepatocytes. Hepatocyte-derived pro-inflammatory markers HMGB1 and IL-33 increase in Huh-7.5 (E) and HepG2 (F) when they are co-cultured with inflammatory producer macrophages. These increases were completely blocked when the target cells were co-cultured with ncRILP-expressing producer inflammatory macrophages. n=3. All data are shown as mean±s.e.m. *P=0.05; **P=0.01; ***P=0.001; ****P=0.0001; ns, not significant (two-tailed unpaired Student's t-test).
Fig 2: EVs are the source of the inflammatory macrophage-derived signal. (A) qPCR analysis of THP-1 cells that were treated with crude conditioned medium, an EV-free soluble fraction or purified EVs (5 µg) for 24 h. Only treatment with crude medium and purified EVs induced an inflammatory response. n=3. (B) No LPS was detected in the EV preparations, assuring that any EV-mediated effect seen was not due to the carry-over of LPS. The amount of LPS in medium alone serves as a positive control. n=4. (C,D) EVs derived from ncRILP-expressing cells are protective. THP-1 (C) or Huh7.5 (D) cells were incubated with cRILP or ncRILP EVs and treated with LPS. qPCR analysis shows that treatment with cRILP EVs increases levels of several inflammatory markers (IL-1ß, IL-6, HMGB1 and IL-33). Treatment with ncRILP EVs protects against LPS-mediated inflammation and significantly reduces the expression of inflammatory markers. Note an increase in the anti-inflammatory marker IL10 with ncRILP. n=3. (E) The EV-mediated crosstalk model was further validated in Huh-7.5 cells by using the EV uptake inhibitor (cytochalasin D, CytD). Cells were treated with CytD (10 µM for 30 min) and then incubated with THP-1 derived EVs. Cell lysates were collected, and IL-33 ELISA was performed. In presence of inhibitor, cRILP EVs no longer induce IL-33 expression in Huh-7.5 cells. These results confirm that EVs are the key regulators of cellular crosstalk. n=3. All data are shown as mean±s.e.m. *P=0.05; **P=0.01; ***P=0.001; ****P=0.0001; ns, not significant (two-tailed unpaired Student's t-test). Ut, untreated.
Fig 3: RILP-mediated regulation of cellular crosstalk in an ex vivo model of animal-derived macrophages. (A) C57BL/6 mice were injected with saline or LPS (0.5 mg/kg body weight). CD11b-enriched primary mouse macrophages were isolated and cultured. IL-1ß secretion from the conditioned medium was measured by ELISA. There is a significant increase in the secretion of IL-1ß from CD11b-enriched primary mouse macrophages that were isolated from LPS-injected mice. n=3. (B,C) CD11b-enriched mouse macrophages isolated from LPS-treated mice also show a significant redistribution of Rab7, thus confirming both inflammasome activation and RILP cleavage in this model. n=a minimum of 30 cells from seven individual experiments. Scale bars: 15 µm. (D) Co-culture of CD11b-enriched primary mouse macrophages and AML12 hepatocytes. CD11b-enriched cells isolated from the LPS-treated mice induce a significant expression of the injury markers CCL2, IL-33 and HMGB1 in target AML12 cells. n=3. (E) Expression of ncRILP–Flag in CD11b-enriched primary mouse macrophages. Figure shows two independent experiments. Western blot for Flag resulted in a non-specific band at ~43 kDa, which is present in all samples, regardless of transfection with empty vector (pCDH-EF1) or ncRILP–Flag. We also detected a band that only reacted with the Flag antibody. This band is present in only the samples transfected with ncRILP–Flag. (F) CD11b-enriched primary macrophages were transfected with ncRILP and co-cultured with AML12 cells. Expression of ncRILP significantly decreased the inflammatory response in the hepatocyte. n=3. All data are shown as mean±s.e.m. *P=0.05; **P=0.01; ****P=0.0001 (two-tailed unpaired Student's t-test).
Fig 4: RILP-mediated regulation of cellular crosstalk in mouse macrophages. (A) RAW–RAW co-culture. Inflammatory markers increase in naïve RAW 264.7 cells when they are co-cultured with LPS-treated producer mouse macrophages. n=3. (B) Co-culture of LPS-treated RAW 264.7 cells with naïve AML12 hepatocytes showing increased expression of hepatocyte-specific cell injury markers within the target hepatocyte. n=3. (C) After co-culturing, AML12 target hepatocytes were collected, and the intracellular protein content was assessed by ELISA. Corresponding increases in HMGB1 and IL-33 are seen. n=3. (D) Caspase-1-mediated RILP cleavage in LPS-treated producer RAW 264.7 cells macrophages was confirmed by measuring the cellular distribution of Rab7. Scale bars: 15 µm. (E) The bar graphs represent an ImageJ analysis measuring the distance the Rab7-positive puncta are from the nucleus. n=a minimum of 30 cells from seven individual experiments. (F) Expression of ncRILP-Flag in RAW 264.7 producer cells showing two independent experiments. Western blot analysis for Flag resulted in a non-specific band at ~43 kDa, which is present in all samples, regardless of transfection with empty vector (pCDH-EF1) or ncRILP–Flag. However, we also detected a band that only reacted with the anti-Flag antibody. This band is present in only the samples transfected with ncRILP–Flag. (G) Hepatocyte-derived pro-inflammatory markers HMGB1 and IL-33 increase in target AML12 cells when they are co-cultured with LPS-treated producer RAW 264.7 cells. These increases are completely blocked when ncRILP is expressed in the inflammatory macrophages. n=3. All data are shown as mean±s.e.m. *P=0.05; **P=0.01; ***P=0.001; ****P=0.0001; ns, not significant (two-tailed unpaired Student's t-test). Ut, untreated.
Supplier Page from Abcam for Mouse IL-33 ELISA Kit