Ablation of Type-1 IFN Signaling in Hematopoietic Cells Confers Protection Following Traumatic Brain Injury

TBI induces type-1 IFN release and downstream STAT3/IRF7 activation in an IFNAR1-dependent manner. A, IFNα and IFNβ mRNA levels are elevated in ipsilateral hemispheres of WT compared with IFNAR1^−/− mice 2 and 24 h following TBI, respectively. Data represent mean± SEM, n=3 per group. *p<0.05, ***p<0.001. B, pSTAT3 immunoreactivity is observed 6 h following TBI in WT but not IFNAR1^−/− neuronal cells (labeled with Fox3) in the ipsilateral hemispheres compared to sham-operated mice. pSTAT3 induction is demonstrated in both WT and IFNAR1^−/− neurons 24h after TBI. C, Six hours after TBI, pSTAT3 expression is increased in WT, but not IFNAR1^−/− mouse astrocytes (labeled with GFAP) in the ipsilateral hemispheres compared to sham-operated controls. pSTAT3 is expressed in both WT and IFNAR1^−/− astrocytes 24 h after TBI. Images are representative of n=3 independent experiments. Scale bar, 50 μm. D IRF7 mRNA levels are elevated in ipsilateral hemispheres of WT compared with IFNAR1^−/− mice 2 h following TBI. Data represent mean± SEM, n=3 per group. **p<0.01.

Absence of IFNAR1 contributes to a smaller infarct volume in mice 24 h after TBI. A Representative 10-µm-thick H&E-stained coronal brain section from a WT and IFNAR1^−/− mouse given TBI. B IFNAR1^−/− mice have significantly reduced infarct volumes compared with WT mice 24 h after TBI. Data represent mean ± SEM. *p<0.0; n=6 animals per group. Scale bar, 2 mm.

IFNAR1^−/− mice display lower levels of proinflammatory cytokines and higher levels of the anti-inflammatory cytokine IL-10 in ipsilateral hemispheres compared to WT mice after TBI. A, IL-1β mRNA levels are elevated at 2, 4, and 24 h after TBI in WT but not IFNAR1^−/− mice. B, IL-6 mRNA levels are elevated 4 h after TBI in both WT and IFNAR1^−/− mice. C, IL-10 mRNA levels are elevated 2 and 4 h after TBI in IFNAR1^−/− compared with WT mice. D, WT mice display elevated IL-1β protein compared with IFNAR1^−/− mice 6 h after TBI. E, IL-6 protein levels are elevated in WT compared with IFNAR1^−/− mice 2 h after TBI. F, IFNAR1^−/− mice display higher levels of IL-10 protein compared to WT mice 2 and 24 h after TBI. Data represent mean ± SEM, n=3 per group. *p<0.05, **p<0.01, ***p<0.001.

IFNAR1^−/− mice exhibit increased GFAP staining compared with WT mice after TBI. A, Representative image of GFAP staining in the ipsilateral hemisphere of WT and IFNAR1^−/− mice 24 h after TBI. Scale bar, 200 μm. B, High-resolution image of GFAP staining in the ipsilateral hemisphere of WT and IFNAR1^−/− mice 24 h after TBI. Image region is outlined in the white box in A. Scale bar, 50 μm. C, Quantification of GFAP staining in TBI mice, using fluorescence intensity values to quantify GFAP levels. Data represent mean ± SEM, n=5 per group. **p<0.01.

Iba-1 levels increase after TBI, and are influenced by type-1 IFN signaling. A, Representative image of Iba-1 staining in the ipsilateral hemisphere of WT and IFNAR1^−/− mice 24 h after TBI. Scale bar, 200 μm. B, High-resolution image of Iba-1 staining in the ipsilateral hemisphere of WT and IFNAR1^−/− mice 24 h after TBI. Image region is outlined in the white box in A. Scale bar, 50 μm. C, Quantification of Iba-1 staining in TBI mice using fluorescence intensity values to quantify Iba-1 levels. Data represent mean ± SEM, n=5 per group. p=0.0537.

IFNAR1^−/− microglia exhibit increased CD206 staining compared with WT mice after TBI. CD206 immunoreactivity is observed following TBI in IFNAR1^−/− mice at a greater level compared with WT mice 24 h after TBI. CD206 immunoreactivity is colabeled with the microglial marker Iba-1.

Pre- and post-treatment with MAR1 decreases infarct volume in WT mice given TBI both 24 h and 7 d after TBI. A, WT mice were treated 1 h before surgery with MAR1 (0.5 mg) or an IgG isotype control (0.5 mg); infarct was calculated 24 h after TBI. Data represent mean ± SEM; *p<0.05, n=3 animals per group. B, WT mice were treated 30 min after TBI with MAR1 or an IgG isotype control; infarct was calculated 24 h after TBI. Data represent mean ± SEM; *p<0.05, n=6 animals per group. C, WT mice were treated 30 min and 2 d after TBI with MAR1 or an IgG isotype control; infarct was calculated 7 d after TBI. Data represent mean ± SEM; *p<0.05, n=8 animals per group.

MAR1-treated mice display reduced type-1 IFN and proinflammatory cytokine secretion following TBI. A, IFNα mRNA levels are elevated in IgG-treated mice, compared to MAR1-treated mice 2 h after TBI. B, IFNβ mRNA levels are elevated in IgG-treated mice compared with MAR1-treated mice 4 h after TBI. C, IL-1β protein levels are elevated in IgG-treated mice, compared to MAR1-treated mice 4 h after TBI. D, IL-6 protein levels are elevated in IgG-treated mice compared with MAR1-treated mice 4 h after TBI. E, IL-10 protein levels are unchanged by MAR1 treatment after TBI. Data represent mean ± SEM, n=3 per group. *p<0.05, ***p<0.001.

MAR1 post-treatment significantly improves behavioral outcome after TBI. Three hours after surgery, TBI mice show significant behavioral impairment compared to sham mice in left hindlimb parameters such as stance–swing ratio, percentage swing in stride, and percentage stance in stride (A–C). Data are presented as values of postinjury–preinjury ratios. Post-TBI administration of MAR1 significantly improves behavioral outcome compared to IgG-treated mice in these parameters (D–F). MAR1 and IgG-treated values are presented as fold-change to TBI. Data represent mean± SEM; *p<0.05, n=10 animals per group.

Blocking type-1 IFN signaling in hematopoietic cells engenders protection following TBI. A, MRI T2 images from bone marrow chimeric mice showing TBI lesion 24 h after injury. WT&cenveo_unknown_entity_wingdings_F0E0;WT mice represent C57BL/6 CD45.1 mice irradiated to abolish hematopoietic cells and reconstituted with BL/6 bone marrow. IFNAR1^−/−&cenveo_unknown_entity_wingdings_F0E0;WT mice represent irradiated C57BL/6 CD45.1 mice reconstituted with IFNAR1^−/− bone marrow. WT&cenveo_unknown_entity_wingdings_F0E0;IFNAR1^−/− mice represent irradiated IFNAR1^−/− mice reconstituted with C57BL/6 CD45.1 bone marrow. Scale bar, 1 mm. B, MRI T2 images from chimeric mice showing TBI lesion 7 d after injury. C, IFNAR1^−/−&cenveo_unknown_entity_wingdings_F0E0;WT mice have significantly reduced infarct volumes 7 d after injury. Data represent mean± SEM; **p<0.01, n=6–8 animals per group.

GFAP immunoreactivity is significantly elevated in IFNAR1^−/−&cenveo_unknown_entity_wingdings_F0E0;WT mice 7 d after TBI. A, Representative tiled images using GFAP immunohistochemistry in chimera groups 7 d after TBI. Scale bar, 200 μm. B, High-resolution image of GFAP staining in the ipsilateral hemisphere of all chimeras 7 d after TBI. Image region is outlined in the white box in A. Scale bar, 50 μm. C, Quantification of GFAP staining in TBI mice, using fluorescence intensity values to quantify GFAP levels. Data represent mean ± SEM; n=3 per group, *p=0.0273.

Iba-1 immunoreactivity is significantly elevated in IFNAR1^−/−&cenveo_unknown_entity_wingdings_F0E0;WT mice 7 d after TBI. A, Representative tiled images using Iba-1 immunohistochemistry in chimera groups 7 d after TBI. Scale bar, 200 μm. B, High-resolution image of Iba-1 staining in the ipsilateral hemisphere of all chimeras 7 d after TBI. Image region is outlined in the white box in A. Scale bar, 50 μm. C, Quantification of Iba-1 staining in TBI mice using fluorescence intensity values to quantify Iba-1 levels. Data represent mean ± SEM; n=3 per group, **p=0.0047.