Research ArticleIMMUNOLOGY

VSIG4 mediates transcriptional inhibition of Nlrp3 and Il-1β in macrophages

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Science Advances  09 Jan 2019:
Vol. 5, no. 1, eaau7426
DOI: 10.1126/sciadv.aau7426
  • Fig. 1 VSIG4 inhibits Nlrp3 and Il-1β transcription in vitro.

    PEMs were isolated from Vsig4−/− mice and their C57BL/6 WT littermates. (A) qRT-PCR analysis of mRNA transcription encoding for NLRP3 inflammasome components (each circle represents an individual mouse). (B) PEMs were treatment without or with lipopolysaccharide (LPS; 2 μg/ml) for 6 hours, and cell extracts were immunoblotted for NLRP3 and IL-1β. (C) RAW264.7 cells were stably infected with Len-Cont. or Len-Vsig4 vectors, cells were further treated without or with LPS (2 μg/ml) for 6 hours, and cell extracts were immunoblotted for NLRP3 and IL-1β. (D) PEMs from WT mice were treated with the VG11 mAbs (50 μg/ml) or isotype IgG1 (50 μg/ml) for 6 hours, cells were further activated by LPS (2 μg/ml) for 6 hours, and cell extracts were immunoblotted for NLRP3 and IL-1β. PEMs were stimulated with LPS (2 μg/ml) for 3 hours and then treated with 1.5 μM ATP or 5 μM nigericin for 45 min, or added with SiO2 (500 μg/ml) for 4 hours. (E) Cell extracts were immunoblotted for caspase-1. (F) ELISA analysis of IL-1β and LDH release in cultured supernatants. PEMs were treated with NLRP3 inflammasome stimuli in the presence of isotype IgG1 mAbs (50 μg/ml) or VG11 mAbs (50 μg/ml). (G) Cell extracts were immunoblotted for caspase-1. (H) ELISA analysis of IL-1β and LHD release in cultured supernatants. PEMs were transfected with dsDNA after 12 hours. (I) Cell extracts were immunoblotted for caspase-1. (J) ELISA analysis of IL-1β and LDH release in cultured supernatants. Error bars indicate SEM. NS, not significant. *P < 0.05 and **P < 0.01 (Student’s t test). Nig, nigericin; Casp-1, caspase-1. Data represent one out of three biological replicates, with three technical replicates each at least.

  • Fig. 2 VSIG4 inhibits the transcription of Nlrp3 and Il-1β via activating the AKT-STAT3-A20 axis.

    PEMs and liver tissues were isolated from Vsig4−/− mice and their C57BL/6 WT littermates. (A) Western blot analysis of A20 (each number represents an individual mouse). RAW264.7 cells were transfected with Len-Cont. or Len-Vsig4. (B) Cell extracts were immunoblotted for the indicated protein. (C) A20 in RAW264.7 cells was silenced by shRNA, cells were further treated with LPS (2 μg/ml) for an additional 3 hours, and cell extracts were immunoblotted for the indicated protein. (D) PEMs were treated with LPS (2 μg/ml) in the presence of NF-κB inhibitor BAY 11-7082 (5 μM), and cell extracts were immunoblotted for the indicated molecules after 6 hours. (E) The transcription of Stat3 in RAW264.7 cells was silenced by shRNA, and cell extracts were immunoblotted for A20 and STAT3. LPS-primed RAW264.7 cells were treated with (F) the STAT3 inhibitor S3I-201 (100 mM) and (G) the JAK2 inhibitor TG101348 (10 μM), and cell extracts were immunoblotted for the indicated molecules after 12 hours. (H) ChIP-qPCR analysis of the enrichment of p-STAT3 binding to the potential binding sites in the A20 promoter region in Vsig4+RAW264.7 cells after 3 hours of LPS (2 μg/ml) administration. (I) Analysis of luciferase activity of A20 gene promoter constructs by luciferase reporter assay. Error bars indicate SEM. NS, not significant. *P < 0.05 and **P < 0.01 (Student’s t test). Data represent one out of three biological replicates, with three technical replicates each at least.

  • Fig. 3 VSIG4 cross-links with MS4A6D to activate the JAK2-STAT3-A20 signaling.

    PEMs from C57BL/6 WT mice were isolated. (A) Immunofluoresence double-staining analysis of the coexpression of VSIG4 and MS4A6D. Arrows indicate positive cells, and blue indicates nuclear 4′,6-diamidino-2-phenylindole (DAPI) staining. Scale bar, 20 μm. (B) PEMs were treated with the VG11 mAbs (100 μg/ml) at indicated times, and cell lysates were used for IP with anti-VSIG4 and blotting with anti-MS4A6D. (C) Ms4a6d–HA (hemagglutinin) and Vsig4-Flag were coexpressed in RAW264.7 cells, cell lysates were collected, and VSIG4/MS4A6D interactions were analyzed by Co-IP experiments. (D) Ms4a6d-HA and Vsig4-Flag were cotransfected in RAW264.7 cells, cells were further triggered by VG11 mAbs (100 μg/ml), and MS4A6D and JAK2 interactions were analyzed by Co-IP experiments. (E) PEMs were isolated and further treated with VG11 mAbs (100 μg/ml) for 12 hours, and cell extracts were immunoblotted for p-STAT3/STAT3 and A20. (F) PEMs were isolated from Ms4a6d−/− and WT mice, cells were further treated with LPS (2 μg/ml) for 0 or 3 hours, the transcription of Nlrp3 and Il-1β was analyzed by qRT-PCR. (G) Ms4a6d in Vsig4+RAW264.7 cells was silenced by shRNA, and cell extracts were immunoblotted for the indicated molecules. (H) Overexpression of Ms4a6d in Vsig4+RAW264.7 cells, cells were further treated with LPS (2 μg/ml) for 6 h, and cell extracts were immunoblotted for p-STAT3/STAT3 and A20. (I) Cell extracts were immunoblotted for A20 from Ms4a6d−/− PEMs that were transfected with different Ms4a6d deletion constructs. (J) Vsig4+RAW264.7 cells were treated with VG11 mAbs (100 μg/ml), MS4A6D protein was pulled down by IP experiments, and Ser phosphorylation (p-Ser) was analyzed by Western blot. (K) Ms4a6d−/− PEMs were transfected with different Ms4a6d mutations, and cell extracts were immunoblotted for A20. Error bars indicate SEM. *P < 0.05 and **P < 0.01 (Student’s t test). Data represent one out of three biological replicates, with three technical replicates each at least.

  • Fig. 4 Vsig4 deficiency promotes EAE deterioration in mice.

    The indicated mice were induced to develop EAE by MOG35~55 peptides and complete Freund's adjuvant (CFA) as described in Materials and Methods. (A) The EAE clinical scores were assessed and compared daily. Data of four independent experiments with similar results were merged. The spinal cords were harvested. (B) Representative morphological sections were stained by HE). Magnifications, ×5 (top) and ×20 (bottom). Arrows indicate the infiltrating leukocytes (n = 10 to 15 per group). The infiltrating cells from spinal cords were isolated and purified. (C) Representative flow cytometry plots for immune cell infiltration with antibodies to the indicated molecules. (D) Absolute numbers of infiltrating cells in the spinal cords were compared (n = 10 to 15 per group). (E) Representative flow cytometry plots for IL-17A– and IFN-γ–secreting CD4+ T cells within the spinal cords (CNS) and spleens. (F) The percentage of IL-17A+CD4+T and IFN-γ+CD4+T cells within the CNS and spleens were compared (n = 10 to 15 per group). (G) Absolute numbers of infiltrating cells IL-17A+CD4+T and IFN-γ+CD4+T cells within spinal cords were compared (n = 5 per group). (H) Representative flow cytometry plots for IL-1β secretion from F4/80+ macrophages within spinal cords. (I) Western blot analysis of NLRP3 and IL-1β levels in spinal cords (each number represents an individual mouse). (J) ELISA analysis of IL-1β in serum (each circle represents an individual mouse). All data shown in (B) to (J) were detected at peak of disease. NH, normal health. Error bars indicate SEM. NS, not significant. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 5 Vsig4−/− mice are resistant to DSS-induced colitis.

    Mice were given 3.5% DSS in their drinking water for 6 days to induce colitis. (A) The survival rate was assessed and compared daily for a total of 20 days. Data of five independent experiments with similar results were merged. (B) Comparative analysis of body weight and disease activity index. Data of four independent experiments with similar results were merged. Mice were euthanized at day 6 of DSS feeding. (C) The colon lengths were calculated and compared. (D) Mice were gavaged with FITC-dextran; these mice were euthanized after 3 hours, and sera were collected to detect the FITC-dextran amounts (each circle represents an individual mouse). (E) H&E staining analysis of histopathological changes (left) and semiquantitative scoring of histopathology (right). The asterisk indicates severe edema/inflammation, the arrows indicate the normal epithelium, and L indicates large lymphoid nodules. Scale bar, 300 μm (n = 15 per group). (F) Colonic homogenates were immunoblotted for the indicated molecules (each number represents an individual mouse). (G) In situ immunofluorescence analysis of IL-1β expression in F4/80+ macrophages. Scale bar, 20 μm. Arrows indicate positive cells, and blue indicates nuclear DAPI staining. (H) ELISA analysis of IL-1β in colonic homogenate. (I) In situ immunofluorescence analysis of PCNA expression and immunohistochemistry analysis of BrdU incorporation. Arrows indicate positive cells, and blue indicates DAPI. Scale bar, 20 μm. (J) Representative flow cytometry plots for CD45+ leukocytes infiltrating colonic tissues (left), and the percentages of CD45+ cells/total cells were compared (n = 10 per group). (K) Representative flow cytometry plots for CD45+F4/80+ macrophages, CD45+Ly6C+ monocytes, and Foxp3+CD4+T cell infiltrating colonic tissues. (L) The percentages of indicated cells within colonic tissues were compared (n = 10 per group). Error bars indicate SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. (A) was analyzed by a log-rank test, and others were compared by Student’s t test. Data presented in (E) to (L) were analyzed at day 6 of DSS treatment.

  • Fig. 6 Agonist VSIG4 improves the progress of EAE.

    The C57BL/6 WT mice were induced to develop EAE by MOG35~55 peptides and CFA as described in Materials and Methods; these mice were also treated with VG11 mAbs (100 μg per mouse per week) or mouse isotype IgG1 antibodies (100 μg per mouse per week). (A) The EAE clinical scores were assessed and compared daily. Data of three independent experiments with similar results were merged. At peak of disease, mice were euthanized. (B) The homogenate of spinal cords was immunoblotted for the indicated molecules (each number represents an individual mouse). (C) The PEMs and sera were collected from EAE mice. ELISA analysis of IL-1β in culture supernatants of PEMs and sera (n = 10 per group). The infiltrating cells from spinal cords were isolated and purified. (D) Representative flow cytometry plots for immune cell infiltration with antibodies to the indicated proteins (left), and the percentages of infiltrating cells were compared (right). (E) Representative flow cytometry plots for IL-17A– and IFN-γ–secreting CD4+ T cells within spinal cords. (F) The percentage and number of these cells were calculated and compared (right). n = 10 per group. Error bars indicate SEM. *P < 0.05, **P < 0.01 (Student’s t test).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaau7426/DC1

    Fig. S1. Liganding VSIG4 by complement C3b results from decreasing NLRP3 and proIL-1β expression in macrophages.

    Fig. S2. The expression of NLRP3 and IL-1β in Vsig4−/− PEMs was not affected by VG11 mAbs.

    Fig. S3. Vsig4 deficiency does not cause spontaneous NLRP3 inflammasome activation and IL-1β secretion.

    Fig. S4. MS4A6D interacts with VSIG4 by using a yeast split-ubiquitin screen.

    Fig. S5. Gating strategies of flow cytometry.

    Fig. S6 Inhibition of NLRP3 activity or blocking IL-1β signaling controls the progress of EAE and colitis.

    Fig. S7. Vsig4−/− mice are resistant to 2.5% DSS-induced colitis.

    Fig. S8. Vsig4−/Il-1R1−/− mice deteriorated DSS-induced colonic damage.

    Fig. S9. Model of VSIG4/MS4A6D signaling complex attenuates NLRP3 inflammasome activation.

    Fig. S10. Creating Ms4a6d−/− mice by CRISPR/Cas9-mediated genome engineering.

    Table S1. The PCR primers used in this study.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Liganding VSIG4 by complement C3b results from decreasing NLRP3 and proIL-1β expression in macrophages.
    • Fig. S2. The expression of NLRP3 and IL-1β in Vsig4−/− PEMs was not affected by VG11 mAbs.
    • Fig. S3. Vsig4 deficiency does not cause spontaneous NLRP3 inflammasome activation and IL-1β secretion.
    • Fig. S4. MS4A6D interacts with VSIG4 by using a yeast split-ubiquitin screen.
    • Fig. S5. Gating strategies of flow cytometry.
    • Fig. S6 Inhibition of NLRP3 activity or blocking IL-1β signaling controls the progress of EAE and colitis.
    • Fig. S7. Vsig4−/− mice are resistant to 2.5% DSS-induced colitis.
    • Fig. S8. Vsig4−/Il-1R1−/− mice deteriorated DSS-induced colonic damage.
    • Fig. S9. Model of VSIG4/MS4A6D signaling complex attenuates NLRP3 inflammasome activation.
    • Fig. S10. Creating Ms4a6d−/− mice by CRISPR/Cas9-mediated genome engineering.
    • Table S1. The PCR primers used in this study.

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