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Systemic Administration of Induced Neural Stem Cells Regulates Complement Activation in Mouse Closed Head Injury Models

View Article: PubMed Central - PubMed

ABSTRACT

Complement activation plays important roles in the pathogenesis of central nervous system (CNS) diseases. Patients face neurological disorders due to the development of complement activation, which contributes to cell apoptosis, brain edema, blood-brain barrier dysfunction and inflammatory infiltration. We previously reported that induced neural stem cells (iNSCs) can promote neurological functional recovery in closed head injury (CHI) animals. Remarkably, we discovered that local iNSC grafts have the potential to modulate CNS inflammation post-CHI. In this study, we aimed to explore the role of systemically delivered iNSCs in complement activation following CNS injury. Our data showed that iNSC grafts decreased the levels of sera C3a and C5a and down-regulated the expression of C3d, C9, active Caspase-3 and Bax in the brain, kidney and lung tissues of CHI mice. Furthermore, iNSC grafts decreased the levels of C3d+/NeuN+, C5b-9+/NeuN+, C3d+/Map2+ and C5b-9+/Map2+ neurons in the injured cortices of CHI mice. Subsequently, we explored the mechanisms underlying these effects. With flow cytometry analysis, we observed a dramatic increase in complement receptor type 1-related protein y (Crry) expression in iNSCs after CHI mouse serum treatment. Moreover, both in vitro and in vivo loss-of-function studies revealed that iNSCs could modulate complement activation via Crry expression.

No MeSH data available.


INSCs modulated complement activation via Crry expression.(a) Representative flow cytometric analysis of Crry and C3 expression in iNSCs (red) and iNSCs (Crry KD, blue) after CHI mouse serum administration. Isotype antibodies were used as controls (black). (b,c) Histograms showed the MFI values of Crry and C3 expression in iNSCs and iNSCs (Crry KD) (n = 6/group; ***P < 0.001). (d) INSCs and iNSCs (Crry KD) were treated with CHI mouse serum for 45 min. Then, cell viability was detected by MTT assay. The viability of iNSCs without CHI mouse serum treatment was considered to be at 100% (n = 3/group; *P < 0.05). (e–g) Representative staining for Crry+ (red, e), C3+ (red, f), and C5b-9+ (red, g) depicted Crry, C3 and C5b-9 expression levels in GFP-labelled iNSCs (green) and iNSCs (Crry KD) (green) after CHI mouse serum administration. Nuclei were counterstained with DAPI (blue). (h–j) Histograms indicated the numbers of GFP+/Crry+ (h), GFP+/C3+ (i), and GFP+/C5b-9+ (j) cells between the two groups after CHI mouse serum administration (n = 6/group; **P < 0.01; ***P < 0.001). Scale bar = 10 μm (5 μm).
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f4: INSCs modulated complement activation via Crry expression.(a) Representative flow cytometric analysis of Crry and C3 expression in iNSCs (red) and iNSCs (Crry KD, blue) after CHI mouse serum administration. Isotype antibodies were used as controls (black). (b,c) Histograms showed the MFI values of Crry and C3 expression in iNSCs and iNSCs (Crry KD) (n = 6/group; ***P < 0.001). (d) INSCs and iNSCs (Crry KD) were treated with CHI mouse serum for 45 min. Then, cell viability was detected by MTT assay. The viability of iNSCs without CHI mouse serum treatment was considered to be at 100% (n = 3/group; *P < 0.05). (e–g) Representative staining for Crry+ (red, e), C3+ (red, f), and C5b-9+ (red, g) depicted Crry, C3 and C5b-9 expression levels in GFP-labelled iNSCs (green) and iNSCs (Crry KD) (green) after CHI mouse serum administration. Nuclei were counterstained with DAPI (blue). (h–j) Histograms indicated the numbers of GFP+/Crry+ (h), GFP+/C3+ (i), and GFP+/C5b-9+ (j) cells between the two groups after CHI mouse serum administration (n = 6/group; **P < 0.01; ***P < 0.001). Scale bar = 10 μm (5 μm).

Mentions: To determine the role of Crry expression in mediating the regulatory effects of iNSCs on complement activation, we carried out a loss-of-function study to knock down (KD) the levels of Crry in iNSCs by Crry-specific shRNA lentiviral particles (Supplementary Figure S1). After CHI mouse serum treatment, Crry expression levels in the iNSC (Crry KD) group were substantially lower than those in the iNSC group (n = 6/group, P < 0.05) (Fig. 4a,b). In contrast, C3 expression levels were markedly higher in the iNSC (Crry KD) group than in the iNSC group (P < 0.05) (Fig. 4a,c).


Systemic Administration of Induced Neural Stem Cells Regulates Complement Activation in Mouse Closed Head Injury Models
INSCs modulated complement activation via Crry expression.(a) Representative flow cytometric analysis of Crry and C3 expression in iNSCs (red) and iNSCs (Crry KD, blue) after CHI mouse serum administration. Isotype antibodies were used as controls (black). (b,c) Histograms showed the MFI values of Crry and C3 expression in iNSCs and iNSCs (Crry KD) (n = 6/group; ***P < 0.001). (d) INSCs and iNSCs (Crry KD) were treated with CHI mouse serum for 45 min. Then, cell viability was detected by MTT assay. The viability of iNSCs without CHI mouse serum treatment was considered to be at 100% (n = 3/group; *P < 0.05). (e–g) Representative staining for Crry+ (red, e), C3+ (red, f), and C5b-9+ (red, g) depicted Crry, C3 and C5b-9 expression levels in GFP-labelled iNSCs (green) and iNSCs (Crry KD) (green) after CHI mouse serum administration. Nuclei were counterstained with DAPI (blue). (h–j) Histograms indicated the numbers of GFP+/Crry+ (h), GFP+/C3+ (i), and GFP+/C5b-9+ (j) cells between the two groups after CHI mouse serum administration (n = 6/group; **P < 0.01; ***P < 0.001). Scale bar = 10 μm (5 μm).
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f4: INSCs modulated complement activation via Crry expression.(a) Representative flow cytometric analysis of Crry and C3 expression in iNSCs (red) and iNSCs (Crry KD, blue) after CHI mouse serum administration. Isotype antibodies were used as controls (black). (b,c) Histograms showed the MFI values of Crry and C3 expression in iNSCs and iNSCs (Crry KD) (n = 6/group; ***P < 0.001). (d) INSCs and iNSCs (Crry KD) were treated with CHI mouse serum for 45 min. Then, cell viability was detected by MTT assay. The viability of iNSCs without CHI mouse serum treatment was considered to be at 100% (n = 3/group; *P < 0.05). (e–g) Representative staining for Crry+ (red, e), C3+ (red, f), and C5b-9+ (red, g) depicted Crry, C3 and C5b-9 expression levels in GFP-labelled iNSCs (green) and iNSCs (Crry KD) (green) after CHI mouse serum administration. Nuclei were counterstained with DAPI (blue). (h–j) Histograms indicated the numbers of GFP+/Crry+ (h), GFP+/C3+ (i), and GFP+/C5b-9+ (j) cells between the two groups after CHI mouse serum administration (n = 6/group; **P < 0.01; ***P < 0.001). Scale bar = 10 μm (5 μm).
Mentions: To determine the role of Crry expression in mediating the regulatory effects of iNSCs on complement activation, we carried out a loss-of-function study to knock down (KD) the levels of Crry in iNSCs by Crry-specific shRNA lentiviral particles (Supplementary Figure S1). After CHI mouse serum treatment, Crry expression levels in the iNSC (Crry KD) group were substantially lower than those in the iNSC group (n = 6/group, P < 0.05) (Fig. 4a,b). In contrast, C3 expression levels were markedly higher in the iNSC (Crry KD) group than in the iNSC group (P < 0.05) (Fig. 4a,c).

View Article: PubMed Central - PubMed

ABSTRACT

Complement activation plays important roles in the pathogenesis of central nervous system (CNS) diseases. Patients face neurological disorders due to the development of complement activation, which contributes to cell apoptosis, brain edema, blood-brain barrier dysfunction and inflammatory infiltration. We previously reported that induced neural stem cells (iNSCs) can promote neurological functional recovery in closed head injury (CHI) animals. Remarkably, we discovered that local iNSC grafts have the potential to modulate CNS inflammation post-CHI. In this study, we aimed to explore the role of systemically delivered iNSCs in complement activation following CNS injury. Our data showed that iNSC grafts decreased the levels of sera C3a and C5a and down-regulated the expression of C3d, C9, active Caspase-3 and Bax in the brain, kidney and lung tissues of CHI mice. Furthermore, iNSC grafts decreased the levels of C3d+/NeuN+, C5b-9+/NeuN+, C3d+/Map2+ and C5b-9+/Map2+ neurons in the injured cortices of CHI mice. Subsequently, we explored the mechanisms underlying these effects. With flow cytometry analysis, we observed a dramatic increase in complement receptor type 1-related protein y (Crry) expression in iNSCs after CHI mouse serum treatment. Moreover, both in vitro and in vivo loss-of-function studies revealed that iNSCs could modulate complement activation via Crry expression.

No MeSH data available.