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. 2013 Jun:54:421-31.
doi: 10.1016/j.nbd.2013.01.016. Epub 2013 Jan 30.

Deletion of macrophage migration inhibitory factor worsens stroke outcome in female mice

L Christine Turtzo  1 , Jun Li, Rebecca Persky, Sharon Benashski, Gillian Weston, Richard Bucala, Venugopal Reddy Venna, Louise D McCullough
Affiliations

Deletion of macrophage migration inhibitory factor worsens stroke outcome in female mice

L Christine Turtzo et al. Neurobiol Dis. 2013 Jun.

Abstract

Sex is an important factor in the response to ischemic insults in both the laboratory and the clinic. Inflammation and cell death are points where sex-specific pathways diverge in stroke, and serum estrogen level status affect the response to inflammation. The cytokine macrophage migration inhibitory factor (MIF) is detrimental in experimental stroke models in male animals. However MIF is known to have sex-specific actions on inflammation and wound healing. The role of MIF in the ischemic female brain has not been evaluated. A transient middle cerebral artery occlusion (MCAO/90min) model was used to induce stroke in male, intact female, and ovariectomized female wildtype (WT) and MIF knockout (KO) mice. Infarct size was quantified 72h after stroke. Protein and cytokine levels were assessed post stroke. Female MIF KO mice had significantly larger strokes compared to WT females (mean hemispheric infarct±SEM: 63%±2% versus 29%±3%; n=8; p<0.05). Ovariectomized female MIF KO mice also had larger infarcts than ovariectomized WT littermates (70%±3% versus 47%±4%; n=11; p<0.05). In males, however, infarct size was equivalent between MIF KO and WT mice (63%±2% versus 67%±3%; n=9; p=0.25). There were no significant differences in cytokine levels at 6h post-infarct between mice of either genotype in brain. MIF KO females displayed more microglial activation (ionized calcium binding adaptor molecule 1 (Iba1) immunofluorescence) after stroke than did WT mice or MIF KO males. The larger infarcts in MIF KO females were associated with an early increase in mitochondrial localization of Jun activation domain-binding protein 1 (JAB1). Loss of MIF exacerbated injury in the female brain after experimental stroke, which was independent of changes in pro-inflammatory cytokine levels. This response is sex-specific, and is in part independent of physiological serum levels of estrogen.

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Conflict of interest statement

Competing interests

R Bucala is a coinventor on patents describing MIF inhibitors. The authors have no other conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Genetic deletion of MIF increases infarct size in female mice, but has no effect in male mice at 72 hours after 90 minutes of transient MCAO
Images A through D show representative TTC staining of infarcted brains at similar locations, with infarcted tissue appearing white and viable tissue appearing red. The graphs labeled E through G depict the quantification of infarct sizes based on TTC analysis. A) WT male (n=8); B) MIF KO male (n=9); C) WT intact female (n=6); D) MIF KO intact female (n=6). E) Male mice (n=8 for WT and 9 for MIF KO); F) Randomly hormonal cycling female mice with intact ovaries (*, **, *** = p<0.05) (n=6 per group); G) Ovariectomized female mice (*, **, *** = p<0.05) (n=9 per group).
Figure 2
Figure 2. Neurological deficit scores in WT and MIF KO mice during cerebral ischemia, and at 6, 24, and 72 hours after 90 minutes of transient cerebral ischemia
The horizontal midline of the box plots indicates the mean, with the top and bottom lines of the boxes mark the maximum and minimum values, respectively. A) Male mice; B) Female mice; (n = minimum of 6 per group).
Figure 3
Figure 3. MIF KO mice do not express MIF protein by Western blot (A) or immunofluorescent staining (B)
A) Western blot of total brain protein from MIF KO and WT intact females at 6 hours post-stroke. B) Immunofluorescence of mouse cerebral cortex for MIF (red) and NeuN (green) (n=3 per group). MIF is present in the cytoplasm of neurons in WT mice, while NeuN is located in neuronal nuclei of both WT and MIF KO mice.
Figure 4
Figure 4. Microglial activation in cerebral cortex is higher in MIF KO female mice at 24 hours after stroke than in WT females, WT males, or MIF KO males, as assessed by Iba-1 staining of cerebral cortex
A) WT female sham; B) WT female stroke; C) MIF KO female sham; D) MIF KO female stroke; E) WT male sham; F) WT male stroke; G) MIF KO male sham; H) MIF KO male stroke; Iba1 (red), NeuN (green), and DAPI (blue) (n= 3 per group). The yellow arrows point to examples of activated microglia in post-stroke cortex. Photographs were taken with a 20× objective at approximately 0.3 mm from Bregma within the infarcted cortex or an anatomically similar area in sham animals. I) Quantification of average number of Iba1-positive cell per field of vision (n=3 per group). The * symbol indicates a statistically different difference (p<0.05) from other groups by one-way ANOVA.
Figure 5
Figure 5. Splenic levels of IL-1β are decreased in ovariectomized female MIF KO mice versus WT
A) IL-1β; B) IL-6; C) TNF-α (n = 3 per group). * = p<0.05.
Figure 6
Figure 6. JAB1 levels are upregulated in the mitochondria of MIF KO versus WT intact female mice at 6 hours post-stroke
A) Fractionated protein from mitochondrial and nuclear fractions from intact females at 6 hours post-stroke (n=6 mice per genotype). The JAB1 protein detected is 38 kDa in size, while mitochondrial marker COXIV is 16 kDa; b) Fractionated protein from mitochondrial fractions from intact females at 24 hours post-stroke (n=6 mice per genotype); C) Quantification by densitometry of changes in the ratio of JAB1 to COXIV protein levels relative to sham in intact females at 6 hours and 24 hours post-stroke; D) Detection of MIF protein in mitochondrial lysates from WT mice by Western blotting with representative densitometry; E) Co-immunoprecipitation of JAB and MIF from WT mitochondrial lysates; F) Densitometry of co-immunoprecipitation shown in E expressed as ratio of stroke to sham. The * symbol indicates statistically significant differences (p<0.05) by one-way ANOVA with post-hoc Tukey analysis. Cyto = cytoplasmic fraction, L = ladder, Mito = mitochondrial fraction, C = surgical sham; St = stroke, F = female; Ovx F = ovariectomized female; M = male; Co-IP = co-immunoprecipitation.
Figure 7
Figure 7. JAB1 plays a critical role in the proposed model of MIF’s sex-specific effects in ischemia
In WT mice, MIF’s binding to JAB1 keeps JAB1 sequestered in an inactive form in the cytosol (A). In MIF KO mice, the absence of MIF allows JAB1 to be active. The previously known role of active JAB1 is to affect transcription by binding to other factors such as SRC-1. In the absence of MIF, after stroke in female MIF KO mic, JAB1 enters the mitochondria (B) as demonstrated in the current study. There JAB1 is proposed to interact with Bcl-Gs and/or other factors to decrease mitophagy, resulting in increased release of cytochome C from damaged mitochondria. The increased levels of cytosolic cytochrome c trigger more apoptosis and larger infarcts via the cell death pathway preferred in females.
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