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. 2021 Jan-Jun:296:100527.
doi: 10.1016/j.jbc.2021.100527. Epub 2021 Mar 9.

Adult astrocytes from reptiles are resistant to proinflammatory activation via sustaining Vav1 expression

Nan Du  1 , Hui Li  1 , Chunshuai Sun  1 , Bingqiang He  1 , Ting Yang  1 , Honghua Song  1 , Yingjie Wang  2 , Yongjun Wang  3
Affiliations

Adult astrocytes from reptiles are resistant to proinflammatory activation via sustaining Vav1 expression

Nan Du et al. J Biol Chem. 2021 Jan-Jun.

Abstract

Adult mammalian astrocytes are sensitive to inflammatory stimuli in the context of neuropathology or mechanical injury, thereby affecting functional outcomes of the central nervous system (CNS). In contrast, glial cells residing in the spinal cord of regenerative vertebrates exhibit a weak astroglial reaction similar to those of mammals in embryonic stages. Macrophage migration inhibitory factor (MIF) participates in multiple neurological disorders by activation of glial and immune cells. However, the mechanism of astrocytes from regenerative species, such as gecko astrocytes (gAS), in resistance to MIF-mediated inflammation in the severed cords remains unclear. Here, we compared neural stem cell markers among gAS, as well as adult (rAS) and embryonic (eAS) rat astrocytes. We observed that gAS retained an immature phenotype resembling rat eAS. Proinflammatory activation of gAS with gecko (gMIF) or rat (rMIF) recombinant protein was unable to induce the production of inflammatory cytokines, despite its interaction with membrane CD74 receptor. Using cross-species screening of inflammation-related mediators from models of gMIF- and rMIF-induced gAS and rAS, we identified Vav1 as a key regulator in suppressing the inflammatory activation of gAS. The gAS with Vav1 deficiency displayed significantly restored sensitivity to inflammatory stimuli. Meanwhile, gMIF acts to promote the migration of gAS through regulation of CXCL8 following cord lesion. Taken together, our results suggest that Vav1 contributes to the regulation of astrocyte-mediated inflammation, which might be beneficial for the therapeutic development of neurological diseases.

Keywords: MIF; Vav1; astrocyte; cell migration; cytokine; immunosuppression; neuroinflammation; reptile; spinal cord.

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Conflict of interest statement

Conflict of interest The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1
Temporal analysis of gMIF expression in the severed cord and localization of CD74 receptor in the gAS. A, western blot analysis of gMIF in the 0.5 cm cord segments at injured sites following tail amputation (n = 10) at 1 day, 3 days, 1 week, and 2 weeks, respectively. B, statistical analysis of (A). C, colocalization of CD74 receptor with GFAP-positive cells detected by immunohistochemistry in the spinal cord of gecko (C) and rat (D). E and F, analysis of CD74 receptor in the primary cultured astrocytes (E) and gecko astrocyte cell line Gsn1 (F). Arrowheads indicated the CD74-positive signals. G, western blots analysis of CD74 in the gAS and Gsn1. Error bars represent the standard deviation (p < 0.05). Scale bars, 100 μm in (C) and 50 μm in the magnification, 500 μm in (D) and 50 μm in the magnification, 50 μm in (E) and (F).
Figure 2
Figure 2
Analysis of gMIF proinflammatory effects on the gAS and rAS.A, western blot analysis of prepared gMIF recombinant protein using anti-His tag antibody. M, marker. B, primary cultured gAS and rAS stained with GFAP and Hoechst 33342 with purity over 92%. C and D, ELISA assay of TNF-α and IL-1β production in gAS (C) and rAS (D) stimulated with gradient gMIF for 24 h. All experiments were carried out in triplicate. Error bars represent the standard deviation (p < 0.05). Scale bars, 100 μm in gAS; 50 μm in rAS.
Figure 3
Figure 3
Analysis of rMIF proinflammatory effects on the gAS and rAS. ELISA assay of TNF-α and IL-1β production in gAS (A) and rAS (B) stimulated with gradient rMIF for 24 h. All experiments were carried out in triplicate. Error bars represent the standard deviation (p < 0.05).
Figure 4
Figure 4
Analysis of LPS effects on the gAS and rAS. ELISA assay of TNF-α and IL-1β production in gAS (A) and rAS (B) stimulated with gradient LPS for 24 h. All experiments were carried out in triplicate. Error bars represent the standard deviation (p < 0.05).
Figure 5
Figure 5
Transcriptome sequencing of gAS following stimulation with or without 2.5 μg/ml gMIF for 12 h and 24 h.A, binding assay of gMIF with CD74 receptor in the gAS using anti-His or -CD74 antibody immunoprecipitation. A/G, protein A plus G-Sepharose beads; Ab-His, anti-His antibody; Ab-CD74, anti-mouse CD74 antibody. B, the bar graph showing the number of upregulated (red) genes and downregulated genes in the gAS following stimulation with 2.5 μg/ml gMIF for 12 h and 24 h. C, integration of DEGs following 2.5 μg/ml gMIF treatment of gAS at 12 h and 24 h, respectively. D, heatmap of integrated DEGs in response to stimulation of gMIF. The color scale shown at the top illustrates the relative expression level of the indicated mRNA across all samples: red denotes expression >0 and blue denotes expression <0.
Figure 6
Figure 6
Cross-species screening for the key mediators in suppressing gMIF-induced inflammation in gAS.A, integration of the unaltered genes in gAS following 2.5 μg/ml gMIF stimulation for 12 h and 24 h and the DEGs in rAS stimulated with 2.0 μg/ml rMIF for 12 h and 24 h, respectively. B, GO annotation of the integrated inflammation-related genes. C, a reconstructed gene network was created using IPA on the basis of the integrated genes involved in inflammatory responses.
Figure 7
Figure 7
Effects of Vav1 on suppression of gMIF-mediated inflammation in gAS. A, interference efficiency of three siRNA oligonucleotides for gecko Vav1 was measured by RT-PCR, and siRNA3 was used for the knockdown experiments. B–D, ELISA assay of TNF-α (B), IL-1β (C) and IL-6 (D) production in the gAS following siRNA3 knockdown for 24 h, then treatment with different concentration of gMIF for 24 h. E, transwell determination of gAS migration following siRNA3 knockdown for 24 h, followed by stimulation with 2.5 μg/ml gMIF for 24 h. F, statistical analysis of (E). G, colocalization of Vav1 with GFAP-positive astrocytes in the spinal cord following gecko tail amputation at 0 day and 3 days, respectively. H, western blot analysis of Vav1 in the 0.5 cm cord segments at injured sites following tail amputation (n = 10) at 1 day, 3 days, 1 week, and 2 weeks, respectively. I, statistical analysis of (H). Error bars represent the standard deviation (p < 0.05). Scale bars, 100 μm in (E) and (G).
Figure 8
Figure 8
Analysis of gMIF-mediated regulators in the gAS based on trancriptome sequencing. A, GO terms of DEGs in gAS following stimulation with or without 2.5 μg/ml gMIF for 12 h and 24 h, respectively. B, a reconstructed gene network was created using IPA on the basis of integrated DEGs.
Figure 9
Figure 9
Effects of gMIF on the migration and proliferation of gAS.A, transwell determination of gAS migration stimulated with 2.5 μg/ml gMIF for 24 h. B, statistical analysis of (A). C, EdU assays of gMIF effects on proliferation of gAS stimulated with 0 to 2.5 μg/ml gMIF for 24 h. D, quantification data as shown in (C). Error bars represent the standard deviation. Scale bars, 100 μm
Figure 10
Figure 10
Determination of promoting effects of CXCL8 on gAS migration under regulation of gMIF.A, RT-PCR analysis of CXCL8 expression in gAS following 2.5 μg/ml gMIF stimulation for 12 h and 24 h, respectively. B, RT-PCR analysis of CXCL8 expression in gAS following 2.5 μg/ml gMIF stimulation for 24 h in the presence or absence of 50 μM 4-IPP, the inhibitor of MIF. C, expression analysis of CXCL8 following 2.5 μg/ml gMIF stimulation for 12 h in the presence of 10 μM SP600125, 10 μM SB203580, or 10 μM PD98059, the inhibitor of JNK, P38, and ERK, respectively. D, interference efficiency of three siRNA oligonucleotides for gecko CXCL8 was measured by RT-PCR. E, transwell determination of gAS migration after CXCL8 siRNA3 knockdown for 24 h, followed by stimulation with 2.5 μg/ml gMIF for 24 h. F, quantification data as shown in (E). Error bars represent the standard deviation. Scale bars, 100 μm.
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