Silencing the lncRNA Maclpil in pro-inflammatory macrophages attenuates acute experimental ischemic stroke via LCP1 in mice

Abstract

Long noncoding RNAs (lncRNA) expression profiles change in the ischemic brain after stroke, but their roles in specific cell types after stroke have not been studied. We tested the hypothesis that lncRNA modulates brain injury by altering macrophage functions. Using RNA deep sequencing, we identified 73 lncRNAs that were differentially expressed in monocyte-derived macrophages (MoDMs) and microglia-derived macrophages (MiDMs) isolated in the ischemic brain three days after stroke. Among these, the lncRNA, GM15628, is highly expressed in pro-inflammatory MoDMs but not in MiDMs, and are functionally related to its neighbor gene, lymphocyte cytosolic protein 1 (LCP1), which plays a role in maintaining cell shape and cell migration. We termed this lncRNA as Macrophage contained LCP1 related pro-inflammatory lncRNA, Maclpil. Using cultured macrophages polarized by LPS, M(LPS), we found that downregulation of Maclpil in M(LPS) decreased pro-inflammatory gene expression while promoting anti-inflammatory gene expression. Maclpil inhibition also reduced the migration and phagocytosis ability of MoDMs by inhibiting LCP1. Furthermore, adoptive transfer of Maclpil silenced M(LPS), reduced ischemic brain infarction, improved behavioral performance and attenuated penetration of MoDMs in the ischemic hemisphere. We conclude that by blocking macrophage, Maclpil protects against acute ischemic stroke by inhibiting neuroinflammation.

Keywords: Inflammatory; Ischemic stroke; lncRNA; macrophages; neuroinflammation.

Figure 1.

MoDMs and MiDMs purified from the ischemic hemisphere have distinct lncRNAs expression profiles. (a) Representative FACS gating strategies. After excluding T cells (CD3⁺), B cells (CD19⁺) and neutrophils (Ly6G⁺) from live singlets, MoDMs and Mi/MiDMs are gated as CD45^hiCD11b⁺ (a) and CD45^lowCD11b⁺ (b), respectively. (b) The heatmap represents RNA deep sequencing results. After ischemia, a total of 73 lncRNAs in MiDMs and MoDMs were identified. Four cell groups were studied: microglia (Mi) in the sham group, microglia in the non-ischemic hemisphere, MiDMs and MoDMs in the ischemic hemisphere. (c) Percentage of lncRNA distributions in MoDMs and MiDMs in the ischemic hemisphere. (d) Six lncRNAs highly expressed in MoDMs were selected for further screening: their names, neighbor genes, and functions are listed. These six lncRNAs are also highlighted in the heatmap. Gm20658 and Gm15628 (highlighted in blue) were expressed in MoDMs but not in MiDMs. Gm15413, E230016k23Rik, F630028O10Rik, 170007M16Rik (highlighted in purple) had higher expressions in MoDMs than in MiDMs after stroke.

Figure 2.

The lncRNA Maclpil (GM15628) expression is linked to the pro-inflammatory macrophage M(LPS) polarization in vitro. Bone marrow-derived monocytes (BMDM) were cultured and differentiated into macrophages, then polarized to pro-inflammatory macrophages by adding LPS in vitro. (a) Real-time qPCR shows the Maclpil and iNOS expression time courses at the transcriptional level in BMDM at 0 h, 1 h, 24 h, and 48 h after LPS addition to the cultured macrophages. The values represent Mean ±SD, one-way ANOVA, *, **, **** p < 0.05, p < 0.01, p < 0.0001, respectively. (b) The effects of Maclpil knockdown by siRNA on inflammatory gene expressions in M(LPS) at 48 h after LPS addition. (a) The RT-qPCR results show that transfecting a Maclpil-specific siRNA in M(LPS) resulted in reductions of Maclpil RNA expressions. Maclpil reduction resulted in the decrease of the pro-inflammatory genes, iNOS and IL-1ß (b–c), but increases in the anti-inflammatory genes, Argnase1 and Fizz1 (e–f). (c) ELISA results show that Maclpil knockdown resulted in the reduction in IL-1β proteins in cell supernatant of cultured M(LPS) measured at 48 h after LPS addition. The experiments were repeated three times. n = 3 in each group, one-way ANOVA, *,**, *** p < 0.05, p < 0.01, p < 0.001.

Figure 3.

Maclpil modulates migration and phagocytosis by its actin-relevant neighbor gene LCP1. (a) RT-PCR results show that when Maclpil was knocked down by siRNA in cultured M(LPS), both Maclpil and LCP1 expressions were inhibited for 48 h after LPS addition. ****, p < 0.0001, one-way ANONVA, n = 3. (b) In vitro transwell cell migration assay. The knockdown of LCP1 or Maclpil decreased the cell migration from the upper chamber to the lower chamber. Mock control (cells with transfection reagent only, without adding siRNA). (c) In vitro cell phagocytosis assay. FACS results showed that when LCP1 or Maclpil was knocked down by siRNA, the numbers of cells phagocytosed FITC⁺ latex beads were significantly reduced. Mock control (cells with transfection reagent only, without adding FITC⁺ beads). *, ***, p < 0.05, p < 0.001, one-way ANOVA.

Figure 4.

IV injection of Maclpil-silenced M(LPS) (MSM) protected against acute ischemic stroke. (a) Schematic diagram of experimental design. Behavioral training started five days before surgery. Cells were injected immediately after MCAO surgery (Day 0). Mice were euthanized for TTC staining on day 3 after stroke onset. Behavior test and neurological scores were tested from day 3 to day 7. (b) Representative TTC staining and the bar graph show the statistical results of infarct sizes as percentages measured at three days after ischemic stroke. n = 6–8 mice/group. (c) Survival curve from day 0 to day 15 after MCAO. n = 10–12 mice/group. (d) Neurological score from day 3 to day 7, n = 10–12 mice/group. One-way ANOVA, *,** p < 0.05, p < 0.01. (e) The behavioral assessment was conducted by using the pole test, showing the average walking time from the top to the bottom of the vertical pole. n = 10–12 mice/group. *, **, **** p < 0.05, p < 0.01, p < 0.0001. Two-way ANOVA followed by Tukey's multiple comparisons test (within each column).

Figure 5.

The effects of MSM adoptive transfer on leukocyte accumulations in the ischemic brain. (a) Confocal immunofluorescent staining for CD11b, CD68, and iNOS, and counter stained with DAPI. The top panel shows a representative brain section stained with cresol/violet, on which three indicated regions are identified for cell number quantification. The middle panel shows the representative staining of CD11b⁺ (red) and CD68⁺ (green), and the bottom panel shows the representative staining of CD11b⁺(red) and iNOS⁺ (green) in vehicle, control siRNA, and MSM groups at 72 h after stroke. The bar graphs in the right show CD68⁺ and iNOS⁺ cell numbers and their percentages in total microglia/macrophage (CD11b⁺ cells). Scale bar: 20 μm. n = 5–6 mice/group. *, **p < 0.05, p < 0.01. (b) FACS analysis of the ischemic brain immune cells. The upper pictures show the representative gating methodology in the brain to identify MoDMs, MiDMs, B cells, CD4 and CD8 T cells. Bar graphs of the statistical results for each cell types. n = 5 mice/group, two-way ANOVA, * p < 0.05, **p < 0.01.