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. 2018 Sep 26;38(39):8484-8495.
doi: 10.1523/JNEUROSCI.2203-17.2018. Epub 2018 Sep 10.

TRPM2 Exacerbates Central Nervous System Inflammation in Experimental Autoimmune Encephalomyelitis by Increasing Production of CXCL2 Chemokines

Masato Tsutsui  1 , Ryo Hirase  1 , Sakie Miyamura  1 , Kazuki Nagayasu  1 , Takayuki Nakagawa  2 , Yasuo Mori  3 , Hisashi Shirakawa  4 , Shuji Kaneko  1
Affiliations

TRPM2 Exacerbates Central Nervous System Inflammation in Experimental Autoimmune Encephalomyelitis by Increasing Production of CXCL2 Chemokines

Masato Tsutsui et al. J Neurosci. .

Abstract

Multiple sclerosis (MS) is a chronic inflammatory disorder of the CNS characterized by demyelination and axonal injury. Current therapies that mainly target lymphocytes do not fully meet clinical need due to the risk of severe side effects and lack of efficacy against progressive MS. Evidence suggests that MS is associated with CNS inflammation, although the underlying molecular mechanism is poorly understood. Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable nonselective cation channel, is expressed at high levels in the brain and by immune cells, including monocyte lineage cells. Here, we show that TRPM2 plays a pathological role in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Knockout (KO) or pharmacological inhibition of TRPM2 inhibited progression of EAE and TRPM2-KO mice showed lower activation of Iba1-immunopositive monocyte lineage cells and neutrophil infiltration of the CNS than WT mice. Moreover, CXCL2 production in TRPM2-KO mice was significantly reduced at day 14, although the severity of EAE was the same as that in WT mice at that time point. In addition, we used BM chimeric mice to show that TRPM2 expressed by CNS-infiltrating macrophages contributes to progression of EAE. Because CXCL2 induces migration of neutrophils, these results indicate that reduced expression of CXCL2 in the CNS suppresses neutrophil infiltration and slows progression of EAE in TRPM2-KO mice. Together, the results suggest that TRPM2 plays an important role in progression of EAE pathology and shed light on its putative role as a therapeutic target for MS.SIGNIFICANCE STATEMENT Current therapies for multiple sclerosis (MS), which mainly target lymphocytes, carry the risk of severe side effects and lack efficacy against the progressive form of the disease. Here, we found that the transient receptor potential melastatin 2 (TRPM2) channel, which is abundantly expressed in CNS-infiltrating macrophages, plays a crucial role in development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE progression was suppressed by Knockout (KO) or pharmacological inhibition of TRPM2; this was attributed to a reduction in CXCL2 chemokine production by CNS-infiltrating macrophages in TRPM2-KO mice, resulting in suppression of neutrophil infiltration into the CNS. These results reveal an important role of TRPM2 in the pathogenesis of EAE and shed light on its potential as a therapeutic target.

Keywords: TRP channel; TRPM2; cxcl2; macrophage; multiple sclerosis; neutrophil.

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Figures

Figure 1.
Figure 1.
EAE is less severe in TRPM2-KO mice. A, Mean clinical scores for WT and TRPM2-KO mice after induction of EAE using MOG35-55 peptide and pertussis toxin (number of mice = 8–10). **p < 0.01 (two-way ANOVA with Tukey's multiple-comparisons test). B, C, Representative images of lumbar spinal cord sections from immunized mice at day 16. Sections were stained with LFB (B) and H&E (C) to visualize demyelination and immune cell infiltration. Dashed boxes indicate the area magnified in the image below. D–G, Representative FluoroMyelin-stained images (D) and quantitative analyses (E) of lumbar spinal cord at day 16. Scale bar, 200 μm in D. Representative electronic microscope images (F) and quantitative analyses (G) of axons in lumbar spinal cord sections at day 21. Scale bar, 10 μm in F. Each data point was collected from a different animal. Four mice were used for statistical analysis. *p < 0.05 (unpaired Student's t test). Data are expressed as the mean ± SEM.
Figure 2.
Figure 2.
Pharmacological blockade of TRPM2 prevents EAE development. A, B, Mean clinical scores for WT (A) and TRPM2-KO (B) mice receiving a daily intraperitoneal injection of the TRPM2 inhibitor miconazole (10 mg/kg) or vehicle (DMSO in saline) from days 16 to 28 (number of mice = 5–10). Note that miconazole caused a significant reduction in the clinical score in WT mice, but had no significant effect in TRPM2-KO mice. C–E. Representative images of lumbar spinal cord sections from immunized WT mice receiving a daily intraperitoneal injection of miconazole or vehicle at day 21. Sections were stained with FluoroMyelin (C), LFB (D), or H&E (E) to visualize demyelination and immune cell infiltration. Scale bar, 200 μm. **p < 0.01 (two-way ANOVA, with Tukey's multiple-comparisons test). Data are expressed as the mean ± SEM.
Figure 3.
Figure 3.
Knocking out TRPM2 has no effect on T-cell infiltration into the spinal cord or on T-cell population in secondary lymphoid organs. A, Representative images of lumbar spinal cord sections from immunized mice at day 21 stained with an anti-CD3 antibody. Scale bar, 200 μm. B, Quantitative analysis of the CD3-immunopositive cells shown in A (number of sections = 3–6). C, Quantitative real-time PCR to examine expression of mRNA encoding helper T-cell lineage-specific transcription factors Tbet (Th1), Gata3 (Th2), Rorc (Th17), and Foxp3 (Treg). The relative amounts of mRNA extracted from lumbar spinal cords (at day 21) were normalized against 18s rRNA (number of spinal cords = 5–6). D, E, Representative FACS data for detection of CD3− (D) and CD4+ cells (E) from the spinal cord of naive, WT EAE, and TRPM2-KO EAE mice at day 21 and cumulative quantitative analysis from three to four mice for each group about CD3+ and CD4+ rate or cell number. Singlet and live cells based on forward and side scatter were analyzed. F–K, Representative FACS data for detection of helper T-cell lineage-specific transcription factors T-bet (F, G; Th1), ROR gamma (t) (H, I; Th17), and Foxp3 (J, K; Treg)+ cells from the lymph node (F, H, J) and the spleen (G, I, K) of WT and TRPM2-KO EAE mice at day 10 and cumulative quantitative analysis from 3 mice for each group about T-bet, ROR gamma (t), and Foxp3 positive rate. Singlet and CD4+ live cells based on forward and side scatter were analyzed. Data are expressed as the mean ± SEM.
Figure 4.
Figure 4.
TRPM2-KO mice exhibit reduced macrophages/microglia activation and reduced neutrophil infiltration during the chronic phase of EAE. A–C, Representative sections of lumbar spinal cord from immunized mice obtained at days 7, 14, 21, and 28. Sections were stained with an anti-Iba1 antibody (A; macrophages/microglia), an anti-Gr1 antibody (B; neutrophils), and an anti-GSTpi antibody (C; oligodendrocytes). Scale bar, 100 μm. D–F, Quantitative analysis of Iba1+ cells (D), Gr1+ cells (E), and GSTpi+ cells (F) (number of mice = 3–10). *p < 0.05, ***p < 0.001 vs WT. Data are expressed as the mean ± SEM.
Figure 5.
Figure 5.
Reduced cytokine production in the spinal cord of TRPM2-KO mice. A, B, Amounts of IL6, IFNγ, IL1β, and CXCL2 in the lumbar spinal cord of WT and TRPM2-KO mice at day 14 (A) and day 21 (B) (number of spinal cords = 4–9). C, D, Quantitative real-time PCR analysis of the relative amounts of Cxcl2 (C) and Trpm2 (D) in the lumbar spinal cord. The amount of mRNA extracted from the lumbar spinal cord at days 14 and 21 was normalized against 18s rRNA (number of spinal cords = 5–6). *p < 0.05, **p < 0.01 vs WT (A–C) or naive (D). Data are expressed as the mean ± SEM.
Figure 6.
Figure 6.
CXCL2 is primarily expressed by Iba1+ cells in the CNS. Representative lumbar spinal cord sections obtained from immunized mice at day 14 and stained with anti-Iba1 (top; macrophages/microglia), anti-GFAP (middle; astrocytes), anti-CD3 antibody (bottom; T cells), and anti-CXCL2 (center) antibodies are shown. Images of anti-CXCL2 antibody staining are shown in the center panels and merged images are shown in the right panels. Scale bar, 20 μm.
Figure 7.
Figure 7.
TRPM2 in BM-derived cells contributes to disease progression of EAE. A, Flow cytometry analysis of BM-derived cells in WT/TRPM2-KO BM chimeric mice. Representative histograms showing GFP+ cells in WT (top, control) and BM chimeric mice (bottom). More than 90% of BM-derived cells in all examined chimeric mice were GFP+. B, Mean clinical scores of BM chimeras WT-BM→WT (open circles), WT-BM→TRPM2-KO (closed circles), TRPM2-KO-BM→WT (open squares), and TRPM2-KO-BM→TRPM2-KO (closed squares) after the induction of EAE (number of mice = 6–12). **p < 0.01 (two-way ANOVA with Tukey's multiple comparison test of WT-BM→/WT vs. TRPM2-KO-BM→/WT; WT-BM→/WT vs. TRPM2-KO-BM→/TRPM2-KO). C, Representative sections of lumbar spinal cord from immunized WT-BM→WT mice obtained at day 14 and stained with an anti-Iba1 antibody (macrophages/microglia) and an anti-CXCL2 antibody. Scale bar, 10 μm.
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