CD13 facilitates immune cell migration and aggravates acute injury but promotes chronic post-stroke recovery

Abstract

Introduction: Acute stroke leads to the activation of myeloid cells. These cells express adhesion molecules and transmigrate to the brain, thereby aggravating injury. Chronically after stroke, repair processes, including angiogenesis, are activated and enhance post-stroke recovery. Activated myeloid cells express CD13, which facilitates their migration into the site of injury. However, angiogenic blood vessels which play a role in recovery also express CD13. Overall, the specific contribution of CD13 to acute and chronic stroke outcomes is unknown.

Methods: CD13 expression was estimated in both mice and humans after the ischemic stroke. Young (8-12 weeks) male wild-type and global CD13 knockout (KO) mice were used for this study. Mice underwent 60 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. For acute studies, the mice were euthanized at either 24- or 72 h post-stroke. For chronic studies, the Y-maze, Barnes maze, and the open field were performed on day 7 and day 28 post-stroke. Mice were euthanized at day 30 post-stroke and the brains were collected for assessment of inflammation, white matter injury, tissue loss, and angiogenesis. Flow cytometry was performed on days 3 and 7 post-stroke to quantify infiltrated monocytes and neutrophils and CXCL12/CXCR4 signaling.

Results: Brain CD13 expression and infiltrated CD13⁺ monocytes and neutrophils increased acutely after the stroke. The brain CD13⁺lectin⁺ blood vessels increased on day 15 after the stroke. Similarly, an increase in the percentage area CD13 was observed in human stroke patients at the subacute time after stroke. Deletion of CD13 resulted in reduced infarct volume and improved neurological recovery after acute stroke. However, CD13KO mice had significantly worse memory deficits, amplified gliosis, and white matter damage compared to wild-type animals at chronic time points. CD13-deficient mice had an increased percentage of CXCL12⁺cells but a reduced percentage of CXCR4⁺cells and decreased angiogenesis at day 30 post-stroke.

Conclusions: CD13 is involved in the trans-migration of monocytes and neutrophils after stroke, and acutely, led to decreased infarct size and improved behavioral outcomes. However, loss of CD13 led to reductions in post-stroke angiogenesis by reducing CXCL12/CXCR4 signaling.

Keywords: Angiogenesis; CD13; CXCL12; CXCR4; Cognitive impairment; Ischemic stroke; Myeloid cells; Stroke recovery; Transmigration.

Fig. 1

CD13⁺ cells increase in mouse and human brains at a delayed time point after ischemic stroke. A A decrease in brain CD13⁺ cell numbers acutely after MCAO and MFI of CD13 after stroke (n = 3–5/gp). B Increase in the brain CD13⁺lectin⁺cells at day 15 post-MCAO (n = 4/gp). C An increase in CD13⁺ percentage area at a subacute time point after stroke in human AIS patients. Magnification 20X, scale bar 50 µm, (n = 8–9). Data presented as mean ± SEM. Two-group comparisons were analyzed by unpaired t-test with Welch’s correction. Three-group data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons test (**p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 2

CD13 impedes acute stroke recovery and promotes neutrophil trans-migration. A No gross anatomical difference in large blood vessels between wild-type and CD13KO naïve mice (n = 3). B Smaller infarcts (n = 5–6/group) and C lower NDS (n = 10–11/group) in CD13KO compared to WT after MCAO. D No difference in cerebral blood flow between CD13 wild type and CD13 knockout (n = 3). E Higher microglia counts in the ipsilateral hemisphere of CD13KO mice after stroke. F No difference in the infiltrated monocytes in the ipsilateral hemisphere of CD13KO versus WT mice. G A decrease in ipsilateral infiltrated neutrophils in the CD13KO mice compared to the WT. n = 5–6/gp. Data presented as mean ± SEM. Two-group comparisons were analyzed by unpaired t-test with Welch’s correction. NDS analyzed by Mann–Whitney test and presented as a median with an interquartile range. Two-way ANOVA with Tukey’s multiple comparisons test was used to analyze flow cytometry data (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

CD13 plays a critical role in post-stroke cognitive recovery at chronic time points. A percentage alterations of Y-maze, escape latency, and the number of incorrect entries on Barnes maze, distance moved on an open field at day 7 post-MCAO. B percentage alterations of Y-maze, escape latency and the number of incorrect entries on Barnes maze, distance moved on an open field at day 28 post-MCAO. Data presented as mean ± SEM. Three-group data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons test. n = 6–13/gp. (*p < 0.05, **p < 0.01)

Fig. 4

CD13 KO mice had increased tissue loss, inflammation, and white matter injury at day 30 post-MCAO. A Ventricle area. B percentage tissue loss at day 30 post-MCAO. C Neuron counts day 30 post-MCAO. D Iba-1 and E GFAP counts in the peri-infarct area. F MBP intensity in CC and G striatum. H increased Iba-1 and I GFAP counts in CC at day 30 post-MCAO. Magnification 20X. Scale bar 50 µm. Data presented as mean ± SEM. Three-group data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons test. n = 6/gp. (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 5

CD13 is involved in post-stroke angiogenesis. A Percentage lectin⁺ area and pericyte coverage in the peri-infarct area. B Avg. vessel length and the total number of endpoints at day 30 post-MCAO. Magnification 20×. Scale bar 50 µm. C BrdU⁺lectin⁺ counts. Magnification 40×. Scale bar 20 µm. Data presented as mean ± SEM. Three-group data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons test. n = 6/gp. (*p < 0.05, **p < 0.01)

Fig. 6

Upregulation of CXCL12/CXCR4 signaling after stroke. A Percentage area CXCL12 in human subjects, n = 8. B Gating strategy for CXCL12 on live cells. C MFI CXCL12. D percentage CXCL12 on live cells. E Ipsilateral and contralateral percentage of CD31⁺ ECs in the wild type and CD13KO mice at day 7 post-MCAO. F Percentage of CD31⁺CXCR4⁺ cells. G MFI quantification of CXCR4 on CD31⁺ECs. Data presented as mean ± SEM. Data were analyzed using the unpaired t-test with Welch’s correction. Three-group data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons test. n = 5–7/gp. (*p < 0.05, **p < 0.01)