TGF-β1 modulates microglial phenotype and promotes recovery after intracerebral hemorrhage - PubMed (original) (raw)

. 2017 Jan 3;127(1):280-292.

doi: 10.1172/JCI88647. Epub 2016 Nov 28.

Che-Feng Chang, Brittany A Goods, Matthew D Hammond, Brian Mac Grory, Youxi Ai, Arthur F Steinschneider, Stephen C Renfroe, Michael H Askenase, Louise D McCullough, Scott E Kasner, Michael T Mullen, David A Hafler, J Christopher Love, Lauren H Sansing

TGF-β1 modulates microglial phenotype and promotes recovery after intracerebral hemorrhage

Roslyn A Taylor et al. J Clin Invest. 2017.

Abstract

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from the rupture of a blood vessel in the brain, leading to a mass of blood within the brain parenchyma. The injury causes a rapid inflammatory reaction that includes activation of the tissue-resident microglia and recruitment of blood-derived macrophages and other leukocytes. In this work, we investigated the specific responses of microglia following ICH with the aim of identifying pathways that may aid in recovery after brain injury. We used longitudinal transcriptional profiling of microglia in a murine model to determine the phenotype of microglia during the acute and resolution phases of ICH in vivo and found increases in TGF-β1 pathway activation during the resolution phase. We then confirmed that TGF-β1 treatment modulated inflammatory profiles of microglia in vitro. Moreover, TGF-β1 treatment following ICH decreased microglial Il6 gene expression in vivo and improved functional outcomes in the murine model. Finally, we observed that patients with early increases in plasma TGF-β1 concentrations had better outcomes 90 days after ICH, confirming the role of TGF-β1 in functional recovery from ICH. Taken together, our data show that TGF-β1 modulates microglia-mediated neuroinflammation after ICH and promotes functional recovery, suggesting that TGF-β1 may be a therapeutic target for acute brain injury.

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Conflict of interest statement

The authors have declared that no conflict of interest exists.

Figures

Figure 1

Figure 1. Microglia downregulate proinflammatory genes by 7 days after ICH.

(A) Multiplex assays (IL-6, CCL2, IL-17, IFN-γ, IL-10, IL-4, IL-13) and ELISA assays (TGF-β1 and BDNF) were performed on brain tissue homogenates of the perihematomal regions of WT mice at baseline and 1, 3, 7, 10, and 14 days after ICH. Individual mice represented as one dot with the line at the median value. n = 4–7 mice/time point. (B) Gating strategy to isolate microglia for cell sorting experiments. Samples were gated on live singlets prior to leukocytes being found by forward and side scatter. Samples were then gated on side scatter, CD45int to separate microglia from peripheral leukocytes and then on CD45int, CD11b+ to obtain microglia. (C) qRT-PCR for Il6 and Ccl2 was performed on cell-sorted microglia from CX3CR1-heterozygous mice at baseline, 12 hours, and 1, 3, 7, and 14 days after ICH. Means graphed with SEM; n = 7–16. Results in A and C were analyzed by ANOVA followed by Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2

Figure 2. Temporal transcriptional analysis of microglia after ICH.

(A) Scatter plots show each sample projected on the first two principal components and are color coded according to time point. Biological replicates cluster closely at each time point. (B) Heatmap of the Z score of genes identified by PCA for each sample. Data were clustered hierarchically in GENE-E using one minus Pearson correlation and complete linkage. Data are colored according to row minimum and maximum. Time points are color coded as in A, and days 10–28 cluster most closely. Each replicate includes microglia from 3 brains, and there are 3 biological replicates per time point. (C) GO biological process enrichment analysis was performed using genes appearing in the resolution phase quadrant of the PCA. TGF-β1 pathways account for the majority of the top 15 GO functions. (D) The network diagram shows each gene identified in the resolution phase, including TGF-β1, as a node, and connections are indicated by function.

Figure 3

Figure 3. Microglial alternative activation is required for functional recovery after ICH.

(A) Microglia were cell sorted from WT and CX3CR1-null BM chimeras 14 days after ICH and then processed for qRT-PCR. CX3CR1-null microglia have a significant reduction in TGF-β1 gene expression compared with WT microglia. Means graphed with SEM; Student’s t test; n = 7–9. (B) Brain sections from WT and CX3CR1-null BM chimeras were stained for CD11b (red) and pSMAD2 (green) 14 days after ICH. Nuclei were stained with DAPI. ×40 images, with inset at ×64. Scale bars: 25 μm. Quantification of percent of pSMAD2+, amoeboid CD11b+ graphed as mean with SD; Student’s t test; n = 4. (C) BM chimeras were cylinder tested 1, 3, 7, and 14 days after ICH. CX3CR1-null chimeras maintain a right forelimb preference 14 days after ICH. Means graphed with SEM. Repeated measures ANOVA followed by Tukey’s post hoc test; n = 10–12. (D) Beam walking test shows that CX3CR1-null BM chimeras do not walk as well on the beam 14 days after ICH. Percentage of mice able versus unable to walk is graphed. Wilcoxon rank sum test; n = 11–13. (E) Forced run test shows that CX3CR1-null BM chimeras do not run as quickly as WT BM chimeras 14 days after ICH. Individual dots represent individual mice, with line at the median; Mann-Whitney U test, n = 10–15. *P < 0.05, ***P < 0.001.

Figure 4

Figure 4. TGF-β1 modulates microglia-mediated neuroinflammation in vitro.

(A) Primary microglia respond to TGF-β1 treatment and signal through pSMAD2/3 after 15 minutes of stimulation in vitro. Representative histogram shown with fluorescence minus one (FMO) (gray), thrombin treated (red), and thrombin + TGF-β1 (blue); n = 3. (B) Thrombin + TGF-β1–treated microglia have a reduction in Il6, Ccl2, and Tnf gene expression and an increase in Tgfbr1 gene expression compared with microglia treated with thrombin alone after 8 hours of stimulation. Means graphed with SEM. n = 5 individual experiments with 3 technical replicates per group, per experiment. unstim, unstimulated. (C) Microglia treated with thrombin + TGF-β1 have reduced TNF production after 8 hours of stimulation. Means graphed with SEM. Representative histograms show FMO (gray), unstimulated (purple), TGF-β1 alone (blue), thrombin (red), and thrombin + TGF-β1 (black). n = 8. (D) TNF and IL-6 ELISA on cell supernatants taken after 24 hours of stimulation. Individual wells are represented by dots, with line at the median; n = 5. All statistical analysis performed by ANOVA, followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Figure 5. TGF-β1 treatment promotes better functional outcomes and modulates microglial IL-6 after ICH.

(A) TGF-β1 treatment led to phosphorylation of SMAD2 in myeloid cells in the brain. Brain sections from PBS- or TGF-β1–treated mice were stained at 24 hours for CD11b (red) and pSMAD2 (green). Nuclei were stained with DAPI. Arrow indicates cluster of pSMAD2+CD11b+ cells. Dotted line depicts edge of hematoma. 40×, with inset at 64×. Scale bars: 25 μm. n = 4–5. (B) Cylinder test shows that TGF-β1–treated mice have better functional outcomes 24 hours after ICH. Repeated measures ANOVA, followed by a Tukey’s post hoc test; n = 11–12. (C) TGF-β1–treated microglia have reduced Il6 gene expression 24 hours after ICH. Means graphed with SEM; Student’s t test; n = 7–9. (D and E) TGF-β1–mediated improvements in behavioral outcomes were confirmed using the collagenase model of ICH. Cylinder test (D) and corner test (E) show TGF-β1–treated mice have better functional outcomes at 3 and 7 days after ICH. Repeated measures ANOVA, followed by Tukey’s post hoc test; n = 9–10. *P < 0.05, ***P < 0.001.

Figure 6

Figure 6. An increase in TGF-β1 plasma levels from 6 to 72 hours after ICH is independently associated with better patient outcomes at 90 days.

Plasma TGF-β1 concentrations were measured from controls and ICH patients at 6 ± 6 and 72 ± 6 hours after onset of ICH. (A) Each control patient’s TGF-β1 plasma level is depicted as a gray dot, with the line indicating the median. Each individual patient’s TGF-β1 plasma level is depicted as a dot, with a line connecting each patient’s 6- and 72-hour data. Those who had a decrease in TGF-β1 plasma are depicted in red. (B) Distribution of patient outcomes on the modified Rankin scale by TGF-β1 response. n = 22 controls and 22 ICH patients.

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