Follistatin-like protein 1 enhances NLRP3 inflammasome-mediated IL-1β secretion from monocytes and macrophages

Abstract

Follistatin-like protein 1 (FSTL-1) is overexpressed in a number of inflammatory conditions characterized by elevated IL-1β. Here, we found that FSTL-1 serum concentration was increased threefold in patients with bacterial sepsis and fourfold following administration of LPS to mice. To test the contribution of FSTL-1 to IL-1β secretion, WT and FSTL-1-deficient mice were injected with LPS. While LPS induced IL-1β in the sera of WT mice, it was low or undetectable in FSTL-1-deficient mice. Monocytes/macrophages, a key source of IL-1β, do not normally express FSTL-1. However, FSTL-1 was found in tissue macrophages after injection of LPS into mouse footpads, demonstrating that macrophages are capable of taking up FSTL-1 at sites of inflammation. In vitro, intracellular FSTL-1 localized to the mitochondria. FSTL-1 activated the mitochondrial electron transport chain, increased the production of ATP (a key activator of the nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome) and IL-1β secretion. FSTL-1 also enhanced transcription of the NLRP3 and procaspase 1 genes, two components of the NLRP3 inflammasome. Adenovirus-mediated overexpression of FSTL-1 in mouse paws led to activation of the inflammasome complex and local secretion of IL-1β and IL-1β-related proinflammatory cytokines. These results suggest that FSTL-1 may act on the NLRP3 inflammasome to promote IL-1β secretion from monocytes/macrophages.

Keywords: Cytokines; Follistatin-like protein 1; Inflammasome; Macrophages; Monocytes.

Figure 1. FSTL-1 is involved in the modulation of the inflammatory response in vivo

(A) DBA/1 mice were injected i.p. with 50 µg of LPS and serum was analyzed by FSTL-1 ELISA. The results are expressed as the mean+SEM, n=4 mice/group from one experiment representative of three performed.*p < 0.05 versus PBS, Mann-Whitney test. (B) DBA/1 mice were injected in the hind footpads with 2.5 µg of LPS and euthanized on day 3 after treatment. RNA from paw tissue was assayed by QRT-PCR for FSTL-1. The graph represents a fold change in mRNA level compared with control. The results are expressed as the mean+SEM, n=4 mice/group from one experiment representative of three performed.*p < 0.01 versus control, Mann-Whitney test. (C) Serum samples from healthy volunteers and patients with sepsis were assayed for FSTL-1 by ELISA. Each circle represents an individual subject. Statistical significance determined by Mann-Whitney test. (D) Schematic representation of the gene knockout strategy. (E) Wild type (WT) and FSTL-1 null (KO) mice were injected i.p. with 200 µg of LPS for 3 h and sera were analyzed by ELISA. Each circle represents an individual mouse. The data shown are from one representative experiment of two performed. *IL-1β level was below the 150 pg/mL detection limit. IL-1β in the sera of unstimulated mice was below the level of detection (data not shown). Statistical significance determined by Mann-Whitney test.

Figure 2. Immunolocalization of FSTL-1 in BMDM and mouse paws

(A) Mouse BMDM were incubated with AF488-FSTL-1 or AF488-ovalbumin (green) for 7 h with or without LPS. Nuclei (blue) were co-stained with Hoechst. (B) Mice were injected in the hind footpads with 2.5 µg of LPS and euthanized on day 3 after treatment. Paws from control and LPS-injected mice were cryosectioned (4 µm) for immunohistochemistry. Anti-FSTL-1 (green); anti-CD11b (red); dual-labeled cells (yellow) are indicated by arrows. Nuclei are visualized by Hoechst fluorescence (blue). (A, B) Slides were imaged using a 63× oil-immersion objective. Scale bar 10 µm. The data shown are from one representative experiment of three performed.

Figure 3. FSTL-1 promotes IL-1β secretion from monocytes and macrophages

(A) Human U937 cells stably transfected with plasmid encoding FSTL-1 or a control plasmid were treated with LPS (200 ng/mL) or differentiated into U937-MΦ with PMA (0.5 mM). (B) FSTL-1-transfected mouse macrophage RAW264.7 cells were stimulated with or without 1 µg/mL LPS for 24 h. (C) U937 monocytes were transfected with FSTL-1 protein, or ovalbumin. Cells were stimulated with PMA (10 ng/mL) and LPS (200 ng/mL). (D) BMDM transfected with FSTL-1 protein (or ovalbumin) were stimulated with LPS (1 ng/mL) for 6 h. ATP (0.5 mM) was added for the final 30 min. Supernatants were assayed for human (A, C) or mouse (B, D) IL-1β by ELISA. (A, B, C, D) The results are expressed as the mean+SEM of triplicate samples from one experiment representative of three performed. *P <0.05 versus the controls, two-tailed Student’s test.

Figure 4. FSTL-1 localizes to the mitochondria, increases mitochondrial respiratory complex activity and ATP synthesis and induces IL-1β secretion

(A) Mouse BMDM were transfected with AF488-FSTL-1 or AF488-ovalbumin (green) and incubated for 7 h. (B) Cells were fixed with PFA, permeabilized with Triton X100 and stained with AF488-labeled proteins. (A, B) Slides were imaged using a 63× oil-immersion objective. Mitochondria (red) and nuclei (blue) were co-stained with Mitotracker Red and Hoechst 33342, respectively. Co-localization of FSTL-1 and mitochondria are shown by the yellow color (merged). Scale bar 10 µm. The data shown are from one representative experiment of three performed. (C) Complex I and III enzymatic activity is decreased in mitochondrial extracts from ST2 knockdown (KD) cells: in-gel assay (upper panel), spectrophotometric assay (lower panel). (D) ATP level in intact U937 cells transfected with plasmid encoding human FSTL-1, or a control plasmid. (E) ATP level in U937 cells transfected with FSTL-1 protein. Ovalbumin was used as a control protein. (F) PMA-differentiated U937 transfectants were pre-treated with rotenone (Rot) for 2 h and incubated for an additional 24 h with or without LPS. Supernatants were assayed for IL-1β by ELISA. (C, D, E, F) The results are expressed as the mean+SEM of triplicate samples from one experiment representative of three performed. *P<0.05 versus the controls, **P<0.05 versus cells without rotenone treatment, two-tailed Student’s test.

Figure 5. FSTL-1 promotes caspase-1 activation in monocytes and macrophages

Human U937 cells stably transfected with plasmid encoding FSTL1 or a control plasmid were treated with LPS (200 ng/mL) or differentiated into U937-MΦ with PMA (0.5 mM). FSTL1-transfected mouse macrophage RAW264.7 cells were stimulated with or without 1 µg/mL LPS. Cells were collected at 6 h and processed for Western blot analysis of caspase-1 (A, B) or caspase-1 activity assay (C, D). (A, B) The data shown are from one representative experiment of three performed. (C, D) The results are expressed as the mean + SD of triplicate samples from one experiment representative of three performed, *P<0.05 versus control cells, two-tailed Student’s test.

Figure 6. FSTL-1 increases the NLRP3 inflammasome expression in monocytes and macrophages

Cells were stimulated as described in the legend to Figure 5, collected at 6 h and processed for real-time PCR (A, B) or Western blot analysis (C, D) of NLRP3 inflammasome components. (A, B) The results are expressed as the mean + SEM of triplicate samples from one experiment representative of three performed, *P<0.05 versus control cells, two-tailed Student’s test. (C, D) The data shown are from one representative experiment of three performed.

Figure 7. FSTL-1 enhances secretion of IL-1β, MCP-1, IL-6 and increases expression of NLRP3 inflammasome components in mouse paws

Paws of healthy male DBA/1 mice were injected with Ad-mFSTL-1 or with a control virus, Ad-BglII. Mice were sacrificed on day 8. (A) Paw homogenates were assayed for IL-1β, MCP-1 and IL-6 by corresponding ELISA. (B) Total RNA was isolated from paws and gene expression was analyzed by real time PCR. Expression levels of a particular gene were normalized to 18S rRNA. (A, B) The results are expressed as the mean+SEM, n=4 mice/group from one experiment representative of two performed.*p < 0.05 versus control, Mann-Whitney test. (C) Total protein was assayed for pro-caspase-1, cleaved caspase-1 (p20), NLRP3 and FSTL-1 by Western blotting. The data shown are from one representative experiment of two performed.