Attenuated inflammatory response in triggering receptor expressed on myeloid cells 2 (TREM2) knock-out mice following stroke - PubMed (original) (raw)

Attenuated inflammatory response in triggering receptor expressed on myeloid cells 2 (TREM2) knock-out mice following stroke

Matthias W Sieber et al. PLoS One. 2013.

Abstract

Background: 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.

Methods and results: 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.

Conclusions: 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Expression of TREM2 and pro-inflammatory cytokine transcripts in C57BL/6 and TREM2-KO mice after stroke.

A. 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. 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. C. 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.

Figure 2

Figure 2. Decreased microglial activation in TREM2-KO mice after stroke.

A. 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. 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. C. 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).

Figure 3

Figure 3. Decreased chemokine and chemokine receptor gene transcription in TREM2-KO mice after stroke.

A. 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. 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.

Figure 4

Figure 4. Fewer CD3-positive T-cells in TREM2-KO mice after stroke.

A. 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. 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.

Figure 5

Figure 5. No change in brain tissue injury in TREM2-KO mice after stroke.

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.

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This project was supported by the European Union (STREP, FP6, ‘Age-dependent inflammatory response after stroke’ – ARGES [LSHB-CT-2006-018936]) and is part of the research program of the Jena centre for systems biology of ageing – Jenage (BMBF 0315581). The Neural Regeneration Group at the University Bonn LIFE & BRAIN Center is supported by the Hertie-Foundation and the Deutsche Forschungsgemeinschaft (KFO177, SFB704, FOR1336). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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