Fig 1: Expression of interleukin-33 (IL-33) and candidate TLRs in SGs from patients with IgG4-RD. A, Expression levels of mRNA for IL-33 in SGs from healthy controls (n = 10), patients with chronic sialadenitis (n = 10), patients with SS (n = 15), and patients with IgG4-RD (n = 15). B, Distribution of IL-33 in SGs from a representative healthy control, patient with chronic sialadenitis, patient with SS, and patient with IgG4-RD. Mayer's hematoxylin (blue) counterstained; bars = 100 µm. C, Correlation between expression levels of IL-33 mRNA and candidate TLRs in SGs from patients with IgG4-RD (n = 15), as determined by Spearman's rank correlation test. D, Schematic illustration of the extraction of CD163+ M2 macrophages stimulated with TLR-7 agonist R848. Cells were cultivated as described in Patients and Methods. PBMC = peripheral blood mononuclear cell. E, Production of IL-33 by CD163+ M2 macrophages stimulated with R848, as determined by enzyme-linked immunosorbent assay. IL-33 levels increased in a concentration-dependent manner. In A and E, bars show the mean ± SD. * = P < 0.05; ** = P < 0.01 by Kruskal-Wallis test. See Figure 1 for other definitions.
Fig 2: Effects of TLR-7 agonist R848 in transgenic mice. A, Detection of GFP in bone marrow–derived macrophages (BMMs) from mTLR-7-/- (knockout [KO]) mice and transgenic (Tg) mice by flow cytometric analysis, after staining with anti-CD11b antibody. At least 2 independent experiments were performed. B, Interleukin-6 (IL-6) and IL-12p40 levels in BMMs from knockout mice and transgenic mice left unstimulated or stimulated with R848. After 24 hours of incubation, IL-6 and IL-12p40 were detected in culture medium by enzyme-linked immunosorbent assay (ELISA). Results are from triplicate wells. At least 2 independent experiments were performed. C, Weight of SMGs, kidneys, lungs, and liver in 8-week-old knockout, wild-type (WT), and transgenic mice treated with topical R848 for 4 weeks (n = 8 per group). D and F, H&E-stained (D) and Masson's trichrome–stained (F) sections of SMGs, pancreas, kidneys, lungs, and liver from representative 8-week-old R848-treated knockout, WT, and transgenic mice. Bars = 100 µm. E and G, Focus score (E) and fibrosis score (G) for each organ in 8-week-old knockout, WT, and transgenic mice left untreated or treated with R848 (n = 8 per group). The fibrosis score was calculated from Masson's trichrome staining as described in Patients and Methods. HPF = high-power field. H, Serial sections of SMGs from a representative 8-week-old R848-treated transgenic mouse, stained with H&E and for IgG1, TLR-7, CD206, CD317, and IL-33. Mayer's hematoxylin (blue) counterstained; bars = 100 µm. I, Serum IgG, IgG1, and IgG2a levels in knockout, WT, and transgenic mice (n = 10 per group) before and after R848 treatment, as determined by ELISA. Symbols represent individual mice; horizontal lines show the mean. J, Serum IL-33 levels, determined by ELISA, in knockout, WT, and transgenic mice left untreated or treated with R848 (n = 10 per group). In B, C, E, G, and J, bars show the mean ± SD. * = P < 0.05; ** = P < 0.01 by Mann-Whitney U test in E and G; by Kruskal-Wallis test in C, I and J. See Figure 1 for other definitions.
Fig 3: 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 4: 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 5: 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.
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