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-1β, 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-1β secretion from the conditioned medium was measured by ELISA. There is a significant increase in the secretion of IL-1β 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.