Abstract
BackgroundPolyriboinosinic-polyribocytidylic acid (polyI:C) triggers a strong innate immune response that mimics immune activation by viral infections. Induction of interferon-induced transmembrane protein 3 (Ifitm3) in astrocytes has a crucial role in polyI:C-induced neurodevelopmental abnormalities. Through a quantitative proteomic screen, we previously identified candidate astroglial factors, such as matrix metalloproteinase-3 (Mmp3) and follistatin-like 1 (Fstl1), in polyl:C-induced neurodevelopmental impairment. Here, we characterized the Ifitm3-dependent inflammatory processes focusing on astrocyte-derived Fstl1 following polyI:C treatment to assess the neuropathologic role of Fstl1.MethodsAstrocytes were treated with PBS (control) or polyI:C (10 μg/mL). The conditioned medium was collected 24 h after the polyI:C treatment and used as astrocyte condition medium (ACM). The expression of Fstl1 mRNA and extracellular Fstl1 protein levels were analyzed by quantitative PCR and western blotting, respectively. For functional studies, neurons were treated with ACM and the effects of ACM on dendritic elongation were assayed. To examine the role of Fstl1, recombinant Fstl1 protein and siRNA for Fstl1 were used. To investigate the expression of Fstl1 in vivo, neonatal mice were treated with vehicle or polyI:C on postnatal day 2 to 6.ResultsACM prepared with polyI:C (polyI:C ACM) contained significantly higher Fstl1 protein than control ACM, but no increase in Fstl1 was observed in polyI:C ACM derived from Ifitm3-deficient astrocytes. We found that the production of Fstl1 involves the inflammatory responsive molecule Ifitm3 in astrocytes and influences neuronal differentiation. In agreement, the levels of Fstl1 increased in the hippocampus of polyI:C-treated neonatal mice. COS7 cells co-transfected with both Fstl1 and Ifitm3 had higher extracellular levels of Fstl1 than the cells transfected with Fstl1 alone. Treatment of primary cultured hippocampal neurons with recombinant Fstl1 impaired dendritic elongation, and the deleterious effect of polyI:C ACM on dendritic elongation was attenuated by knockdown of Fstl1 in astrocytes.ConclusionsThe extracellular level of Fstl1 is regulated by Ifitm3 in astrocytes, which could be involved in polyI:C-induced neurodevelopmental impairment.
Highlights
Polyriboinosinic-polyribocytidylic acid triggers a strong innate immune response that mimics immune activation by viral infections
We previously reported that follistatin-like 1 (Fstl1) was a candidate molecule responsible for polyribocytidylic acid (polyI):C-induced neurodevelopmental impairment, and that depletion of interferon-induced transmembrane protein 3 (Ifitm3) in astrocytes attenuated polyI:C astrocyte-conditioned medium (ACM)-induced neurodevelopmental impairment [17, 18]
We compared changes in Fstl1 protein levels between WT and Ifitm3 KO astrocytes after polyI:C treatment (Fig. 1a)
Summary
Polyriboinosinic-polyribocytidylic acid (polyI:C) triggers a strong innate immune response that mimics immune activation by viral infections. Induction of interferon-induced transmembrane protein 3 (Ifitm3) in astrocytes has a crucial role in polyI:C-induced neurodevelopmental abnormalities. Through a quantitative proteomic screen, we previously identified candidate astroglial factors, such as matrix metalloproteinase-3 (Mmp3) and follistatin-like 1 (Fstl1), in polyl:C-induced neurodevelopmental impairment. Much evidence suggests that reactive astrocytes induced by inflammatory cytokines are involved in neurodevelopmental disorders [7, 8], the underlying molecular mechanism is largely unknown. We have reported that Ifitm expression in astrocytes is associated with neurodevelopmental impairment and contributes to brain dysfunction in mice that received daily polyI:C injection from postnatal day 2 to day 6 [16]. We found that humoral factors released from polyI:C-treated astrocytes have crucial roles in Ifitm3-dependent impairment of neuronal maturation of cultured neurons [17]. In addition to Mmp, we have identified several candidates of humoral factors including follistatin like-1 (Fstl1) by proteomic analysis [18]
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