Attenuated Inflammatory Response in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Knock-Out Mice following Stroke

<div><h3>Background</h3><p>Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial surface receptor involved in phagocytosis. Clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. The role of TREM2 following stroke is currently unclear.</p> <h3>Methods and Results</h3><p>As an experimental stroke model, the middle cerebral artery of mice was occluded for 30 minutes with a range of reperfusion times (duration of reperfusion: 6 h/12 h/24 h/2 d/7 d/28 d). Quantitative PCR (qPCR) revealed a greatly increased transcription of TREM2 after stroke. We subsequently analyzed the expression of pro-inflammatory cytokines, chemokines and their receptors in TREM2-knockout (TREM2-KO) mice via qPCR. Microglial activation (CD68, Iba1) and CD3-positive T-cell invasion were analyzed via qPCR and immunohistochemistry. Functional consequences of TREM2 knockout were assessed by infarct volumetry. The acute inflammatory response (12 h reperfusion) was very similar between TREM2-KO mice and their littermate controls. However, in the sub-acute phase (7 d reperfusion) following stroke, TREM2-KO mice showed a decreased transcription of pro-inflammatory cytokines TNFα, IL-1α and IL-1β, associated with a reduced microglial activity (CD68, Iba1). Furthermore, TREM2-KO mice showed a reduced transcription of chemokines CCL2 (MCP1), CCL3 (MIP1α) and the chemokine receptor CX3CR1, followed by a diminished invasion of CD3-positive T-cells. No effect on the lesion size was observed.</p> <h3>Conclusions</h3><p>Although we initially expected an exaggerated pro-inflammatory response following ablation of TREM2, our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in TREM2-KO mice. We therefore conclude that TREM2 appears to sustain a distinct inflammatory response after stroke.</p> </div

Expression of TREM2 and pro-inflammatory cytokine transcripts in C57BL/6 and TREM2-KO mice after stroke.

<p><b>A.</b> Gene transcripts for TREM2 were determined at 6 h, 12 h, 24 h, 2 d, 7 d and 28 d after stroke in the ipsilateral ischemic hemisphere of C57BL/6 mice. TREM2 was up-regulated at 7 d and 28 d after stroke. Data are displayed as mean ± s.e.m. (ratio ipsi vs. contra), n = 4 each, ***p≤0.001. <b>B.</b> TREM2 gene transcripts were determined at 12 h, 7 d and 28 d after stroke (MCAO) as well as after sham procedure (Sham) in TREM2-KO mice (KO) and littermate controls (WT). Increased TREM2 gene transcription was detected in the ischemic hemisphere of littermate controls (WT MCAO ipsi) at 7 d and 28 d. No increased gene transcripts for TREM2 were observed after stroke in littermate controls at the contralateral side (WT MCAO contra) and following sham procedure (WT Sham). TREM2-KO mice (KO) did not show any TREM2 gene transcription. <b>C.</b> Gene transcripts of cytokines were determined after stroke (MCAO) as well as after sham procedure (Sham) in TREM2-KO mice (KO) and littermate controls (WT). A strong up-regulation of pro-inflammatory cytokines (TNFα, IL-1α, IL-1β and IL-6) was observed 12 h after stroke. A reduced gene transcription of TNFα, IL-1α and IL-1β was observed in TREM2-KO (KO MCAO ipsi) compared to littermate control mice (WT MCAO ipsi) at 7 d after stroke. No differences in cytokine gene transcription were revealed between TREM2-KO mice (KO MCAO ipsi) and littermate controls (WT MCAO ipsi) at 12 h and at 28 d. No increased cytokine expression was observed after stroke in littermate controls at the contralateral side (contra) and in sham operated mice (Sham). Bars represent mean ± s.e.m. (mRNA copies per 1,000 Gapdh), sham: n = 3 each and MCAO: n = 5/6 each, WT ipsi vs. WT contra ^#p≤0.05, ^##p≤0.01, WT ipsi vs. KO ipsi *p≤0.05, **p≤0.01, ***p≤0.001.</p

Fewer CD3-positive T-cells in TREM2-KO mice after stroke.

<p><b>A.</b> CD3-positive T-cells invaded the infarct core at 7 d and 28 d following stroke. Increased numbers of CD3-positive T-cells were visible in the infarct core 28 d after stroke in littermate controls, but not in TREM2-KO mice. Scale bar 50 µm. <b>B.</b> Fewer CD3-positive T-cells were detected 28 d after stroke in TREM2-KO mice compared to their littermate controls. Bars represent mean ± s.e.m, n = 3 each, WT ipsi vs. KO ipsi *p≤0.05.</p

Decreased microglial activation in TREM2-KO mice after stroke.

<p><b>A.</b> Post-ischemic Iba1 positive cells (green) displayed the typical activated amoeboid phenotype in the glial scar and infarct core at 7 d and 28 d in WT mice, whereas they remained in a ramified phenotype in TREM2-KO mice. Nuclei of cells counterstained with DAPI (for a better distinction the color was changed to red). Scale bar 20 µm. <b>B.</b> Fewer Iba1 positive activated microglial cells were revealed in the glial scar of TREM2-KO mice (KO) at 7 d following stroke compared to littermate controls (WT). Bars represent mean ± s.e.m., n = 5/6 each, WT ipsi vs. KO ipsi ***p≤0.001. <b>C.</b> Gene transcripts for Iba1 and CD68 were determined at 12 h, 7 d and 28 d after stroke in TREM2-KO mice (KO) and littermate controls (WT). Attenuated gene transcription of Iba1 and CD68 was observed 7 d after stroke in TREM2-KO (KO) mice compared to control (WT) mice. Bars represent mean ± s.e.m. (mRNA copies per 1,000 Gapdh), n = 5/6 each, WT ipsi vs. KO ipsi *p≤0.05 (Iba1), ⁺p≤0.05 (CD68).</p

No change in brain tissue injury in TREM2-KO mice after stroke.

<p>Infarct volumes of TREM2-KO and littermate control mice were analyzed at 7 d and 28 d based on Map2 immunohistochemistry. TREM2-KO mice and littermate control mice showed the same relative loss in Map2 immunostaining as a sign of tissue injury. Box and whisker plots represent mean ± s.e.m. (mm³ injured tissue vs. ipsilateral hemisphere in %), n = 9–14 each.</p

Decreased chemokine and chemokine receptor gene transcription in TREM2-KO mice after stroke.

<p><b>A.</b> The mRNAs for the chemokines CCL3, CCL2 and CCL5 were increased following stroke in littermate controls (WT) as well as in TREM2-KO mice, with a peak at 12 h for CCL3 and CCL2 and at 7 d for CCL5. Reduced gene transcription of CCL3 and CCL2 was observed at 7 d after stroke in TREM2-KO mice compared to littermate controls. No increased chemokine expression was observed after stroke in littermate controls at the contralateral side and in sham-operated mice. <b>B.</b> Gene transcripts of chemokine receptors were increased after stroke, with a peak at 12 h for CCR1 and at 7 d for CCR2, CCR5 and CX3CR1. Chemokine receptor CX3CR1 expression was attenuated following stroke in TREM2-KO mice. Post-ischemic expression of chemokine receptors was unchanged in littermate controls at the contralateral side and in sham-operated mice. Bars represent mean ± s.e.m. (mRNA copies per 1,000 Gapdh), sham: n = 3 each and MCAO: n = 5/6 each, WT ipsi vs. WT contra ^#p≤0.05, ^##p≤0.01, ^###p≤0.001, WT ipsi vs. KO ipsi *p≤0.05, **p≤0.01.</p

Additional file 1: Figure S1. of Age-related macular degeneration associated polymorphism rs10490924 in ARMS2 results in deficiency of a complement activator

Expression vector with ARMS2 coding sequence. (PDF 268 kb