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. 2017 Dec 1;313(6):L1069-L1086.
doi: 10.1152/ajplung.00353.2017. Epub 2017 Sep 14.

Versican is produced by Trif- and type I interferon-dependent signaling in macrophages and contributes to fine control of innate immunity in lungs

Mary Y Chang  1 , Inkyung Kang  2 , Michael Gale Jr  3 , Anne M Manicone  4 , Michael G Kinsella  2 , Kathleen R Braun  2 , Tara Wigmosta  1 , William C Parks  4   5 , William A Altemeier  4 , Thomas N Wight  2 , Charles W Frevert  6   4
Affiliations

Versican is produced by Trif- and type I interferon-dependent signaling in macrophages and contributes to fine control of innate immunity in lungs

Mary Y Chang et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

Growing evidence suggests that versican is important in the innate immune response to lung infection. Our goal was to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. We first defined the signaling events that regulate versican expression, using bone marrow-derived macrophages (BMDMs) from mice lacking specific Toll-like receptors (TLRs), TLR adaptor molecules, or the type I interferon receptor (IFNAR1). We show that LPS and polyinosinic-polycytidylic acid [poly(I:C)] trigger a signaling cascade involving TLR3 or TLR4, the Trif adaptor, type I interferons, and IFNAR1, leading to increased expression of versican by macrophages and implicating versican as an interferon-stimulated gene. The signaling events regulating versican are distinct from those for hyaluronan synthase 1 (HAS1) and syndecan-4 in macrophages. HAS1 expression requires TLR2 and MyD88. Syndecan-4 requires TLR2, TLR3, or TLR4 and both MyD88 and Trif. Neither HAS1 nor syndecan-4 is dependent on type I interferons. The importance of macrophage-derived versican in lungs was determined with LysM/Vcan-/- mice. These studies show increased recovery of inflammatory cells in the bronchoalveolar lavage fluid of poly(I:C)-treated LysM/Vcan-/- mice compared with control mice. IFN-β and IL-10, two important anti-inflammatory molecules, are significantly decreased in both poly(I:C)-treated BMDMs from LysM/Vcan-/- mice and bronchoalveolar lavage fluid from poly(I:C)-treated LysM/Vcan-/- mice compared with control mice. In short, type I interferon signaling regulates versican expression, and versican is necessary for type I interferon production. These findings suggest that macrophage-derived versican is an immunomodulatory molecule with anti-inflammatory properties in acute pulmonary inflammation.

Keywords: hyaluronan synthase 1; inflammation; type I interferons; versican, syndecan-4.

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Figures

Fig. 1.
Fig. 1.
Role of TLR signaling molecules in LPS-mediated regulation of versican in macrophages. BMDMs from WT, TLR2−/−, TLR4−/−, or TLR2/4−/− (A) and WT, MyD88−/−, or Trifmutant (B) mice were treated with PBS, LPS (10 ng/ml), LPSEB (10 ng/ml), or Pam3Csk (10 μg/ml) for 4 h, as indicated (BMDMs from n = 3–7 mice per group). *P < 0.05, **P < 0.01, ****P < 0.0001.
Fig. 2.
Fig. 2.
Role of TLR signaling molecules in LPS-mediated regulation of HAS1 in macrophages. BMDMs from WT, TLR2−/−, TLR4−/−, or TLR2/4−/− (A) and WT, MyD88−/−, or Trifmutant (B) mice were treated with PBS, LPS (10 ng/ml), LPSEB (10 ng/ml), or Pam3Csk (10 μg/ml) for 4 h, as indicated (BMDMs from n = 3–7 mice per group). **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 3.
Fig. 3.
Role of TLR signaling molecules in LPS-mediated regulation of syndecan-4 in macrophages. BMDMs from WT, TLR2−/−, TLR4−/−, or TLR2/4−/− (A) and WT, MyD88−/−, or Trifmutant (B) mice were treated with PBS, LPS (10 ng/ml), LPSEB (10 ng/ml), or Pam3Csk (10 μg/ml) for 4 h, as indicated (BMDMs from n = 3–7 mice per group). **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 4.
Fig. 4.
Role of type I interferons and the type I interferon receptor in LPS-mediated regulation of versican, HAS1, and syndecan-4. A: BMDMs from WT, TLR4−/−, or Trifmutant mice were treated with PBS or LPS (10 ng/ml), as indicated. B: WT BMDMs were treated with PBS, IFN-α, -β, or -γ (100 and 1,000 U/ml), or LPS (10 ng/ml), as indicated. C–E: BMDMs from WT, Trifmutant, or Ifnar1−/− mice were treated with PBS, IFN-α, -β, or -γ (100 U/ml), or LPS (10 ng/ml), as indicated. All treatments were for 4 h (BMDMs from n = 3 mice per group). *P < 0.05, ***P < 0.001, ****P < 0.0001.
Fig. 5.
Fig. 5.
Involvement of TLR4, Trif, Ifn-β, and Ifnar1 in poly(I:C)-mediated regulation of versican, HAS1, and syndecan-4. A: BMDMs from WT, TLR4−/−, or Trifmutant mice were treated with PBS or poly(I:C) (10 or 100 μg/ml) for 4 h, as indicated. B–D: BMDMs from WT, TLR4−/−, Trifmutant, or Ifnar1−/− mice were treated with PBS or poly(I:C) (10 or 100 μg/ml) for 4 h, as indicated. (BMDM from n = 3–5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 6.
Fig. 6.
Effects of signaling inhibitors on versican, HAS1, and syndecan-4 expression. BMDMs from WT mice were pretreated with DMSO vehicle or 50 μM LY294002 (A–C) or 50 ng/ml rWnt3a, 30 mM LiCl, 5 μM CHIR99021, 5 μM XAV939, or 10 μM ICG001 (D) for 30–60 min before treatment with PBS or LPS (10 ng/ml) for 4 h, as indicated (BMDMs from n = 3 mice per group). **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 7.
Fig. 7.
Targeting strategy for generation of Vcanfl/fl mice and characterization of macrophages from LysM/Vcan−/− mice. A: unstimulated BMDMs from WT (Vcanfl/fl), LysM+/0/Vcan−/−, or LysM/Vcan−/− mice were evaluated for basal levels of versican mRNA. B: BMDMs from WT (Vcanfl/fl), LysM+/0/Vcan−/−, or LysM/Vcan−/− mice were treated with PBS or LPS (10 ng/ml) for 4 h, as indicated. C: BMDMs from WT (Vcanfl/fl) or LysM/Vcan−/− mice were treated with PBS or poly(I:C) (100 μg/ml) for 4 h, as indicated. D: BMDMs from WT (Vcanfl/fl), LysM+/0/Vcan−/−, or LysM/Vcan−/− mice were treated with PBS or LPS (10 ng/ml) for 24 h, as indicated. Secreted proteoglycans were purified from the media and analyzed by Western immunoblotting. E: alveolar macrophages from WT or LysM/Vcan−/− mice were treated with PBS or LPS (10 ng/ml) for 4 h, as indicated. BMDMs or alveolar macrophages from n = 3 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 8.
Fig. 8.
Cytokine and chemokine production by LysM/Vcan−/− macrophages. Fold changes in the mRNA from cell lysates (A, C, E, G) and amount of cytokines and chemokines from supernatant (B, D, F, H) of BMDMs from control (Vcanfl/fl) and LysM/Vcan−/− mice 24 h after treatment with poly(I:C) (10 and 100 ng/ml) (BMDMs from n = 3 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 9.
Fig. 9.
Cellular recruitment into air space of lungs of LysM/Vcan−/− mice. WT and LysM/Vcan−/− mice were exposed to oropharyngeal treatment with poly(I:C) (50 μg/mouse). Bronchoalveolar lavage was performed after 24 h, and differential cell counts were analyzed in lavage fluid. Values are shown for total (A), mononuclear (B), and polymorphonuclear (C) cells (n = 7–13 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 10.
Fig. 10.
Recovery of cytokines and chemokines in lungs of LysM/Vcan−/− mice. WT and LysM/Vcan−/− mice were exposed to oropharyngeal treatment with poly(I:C) (50 μg/mouse). A: IFN-β. B: IL-10. C: CCL2/MCP-1. D: CXCL2/MIP2. E: IL-6. F: TNF-α. G: CXCL1/KC. Bronchoalveolar lavage was performed after 24 h, and ELISAs were performed on cell-free lavage fluid (n = 3–7 mice per group). *P < 0.05, **P < 0.01.
Fig. 11.
Fig. 11.
Schematic depicting pathways by which LPS and poly(I:C) regulate expression of versican, HAS1, and syndecan-4. Engagement of macrophage Toll-like receptors TLR4 and TLR3 by LPS and poly(I:C), respectively, result in enhanced versican expression. Subsequent to activation of TLR4 and TLR3, engagement of the TRIF adaptor molecule is known to activate transcription factors IRF3/7 that lead to production of type I interferons (IFN-α/-β) and recognition by type I interferon receptors (IFNAR1/2). Signaling events downstream of IFNAR activation lead to production of versican; the transcription factor(s) mediating expression of versican in this response is still to be determined. In contrast, expression of both HAS1 and syndecan-4 result from TLR4- and TLR2-mediated engagement of the MyD88 adaptor molecule. In addition, syndecan-4 expression results from TLR4- and TLR3-mediated engagement of TRIF and downstream signaling events that are independent of type I IFNs.
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