Hypoxia Inducible Factor-1α in Astrocytes and/or Myeloid Cells Is Not Required for the Development of Autoimmune Demyelinating Disease

HIF-1α is expressed in astrocytes and microglia/macrophages in the spinal cord of EAE mice. A, Immunohistochemical staining for HIF-1α in spinal cord sections from healthy mice and at peak EAE, 17 d p.i. Absorption control after anti-HIF-1α antibody incubation with HIF-1α blocking peptide at peak EAE. Scale bar, 300 μm. Arrows indicate neuronal cells. B, Higher-magnification of HIF-1α immunostaining in the grey and white matter of the spinal cord of healthy and EAE mice. Scale bar, 50 μm. Arrows indicate neuronal cells. C, Double-immunofluorescence with antibodies against HIF-1α (green) and GFAP (top) or isoB4 (bottom) (red) of spinal cord sections of EAE mice 17 d after immunization. Scale bar, 50 μm. Arrows indicate HIF-1α⁺isoB4^– cells with astrocyte-like morphology (green).

Genetic depletion or overexpression of HIF-1α in myeloid cells does not affect the clinical course of EAE, demyelination, and inflammatory infiltrates. A, Clinical scores of mice genetically depleted for HIF-1α in myeloid cells lysM-Cre:HIF-1α^fl/fl (n = 17) and control mice HIF-1α^fl/fl (n = 22). B, Clinical scores of mice overexpressing HIF-1α in microglia/macrophages lysMCre:VHL^fl/fl (n = 20) and control mice VHL^fl/fl (n = 15). A and B, For each genotype, day of onset, maximum clinical score, and percentage of mice developing signs of paralysis (score ≥3) were calculated using mixed linear effects models, and the results are presented in Table 1. Data are presented as mean ± SEM. C, LFB/PAS staining shows demyelination in HIF-1α^fl/fl (n = 5) and lysM-Cre:HIF-1α^fl/fl (n = 5). Data are presented as mean ± SEM. Statistical analysis was performed using the unpaired t test (p = 0.28 ^l ). D, Immunohistochemistry for CD3 shows no significant differences in inflammation between HIF-1α^fl/fl (n = 5) and lysM-Cre:HIF-1α^fl/fl (n = 5). Data are expressed as number of CD3+ cells/mm² per mouse and presented as mean ± SEM. Statistical analysis was performed using the unpaired t test (p = 0.14 ^m ). E, Immunohistochemistry for Mac-2 shows no significant differences in inflammation between HIF-1α^fl/fl (n = 4) and lysM-Cre:HIF-1α^fl/fl (n = 4). Data are expressed as percentage of Mac-2+ area per mouse and presented as mean ± SEM. Statistical analysis was performed using unpaired t test (p = 0.153 ⁿ ). Superscript letters refer to the statistical results in Tables 1, 2, and Results.

Genetic depletion of HIF-1α in astrocytes decreases expression of HIF-1α and iNOS in astrocytes. A, Staining for HIF-1α in GFAP-Cre:HIF-1α^fl/fl and HIF-1α^fl/fl mice after EAE. B, Quantification of iNOS expression in Mac-2+ macrophages and GFAP-expressing astrocytes at peak EAE in HIF-1α^fl/fl mice (n = 5). Area = 0.056 mm². Data are presented as mean ± SEM from n = 5 mice. Statistical analysis was performed using an unpaired t-test with Welch’s correction (p = 0.003 ^° ). C, Quantification iNOS+ cells at peak EAE in HIF-1α^fl/fl (n = 5) and GFAP-Cre:HIF-1α^fl/fl (n = 4) mice. Area = 0.056 mm². Data are presented as mean ± SEM. (continued on page 10). Statistical analysis was performed an unpaired t test (p = 0.034 ^p ). D, Double-immunofluorescence of GFAP (green) and iNOS (red) in HIF-1α^fl/fl and GFAP-Cre:HIF-1α^fl/fl mice at peak EAE. Statistical analysis was performed using an unpaired t test (p = 0.01 ^q ). E, Quantification of iNOS+ GFAP+ cells reveals reduced iNOS-expressing astrocytes in GFAP-Cre:HIF-1α^fl/fl (n = 4) compared with HIF-1α^fl/fl (n = 5) mice. Area = 0.056 mm². Statistical analysis was performed using an unpaired t test (p = 0.0159 ^q ). F, Quantification of GFAP+ astrocytes shows similar numbers of astrocytes in GFAP-Cre:HIF-1α^fl/fl (n = 4) and HIF-1α^fl/fl (n = 5) mice at peak EAE. Area = 0.056 mm². Data are presented as mean ± SEM. Statistical analysis was performed using an unpaired t test (p = 0.06 ^r ). Superscript letters refer to the statistical results in Tables 1, 2, and Results.