β1-Integrin– and KV1.3 channel–dependent signaling stimulates glutamate release from Th17 cells
Katharina Birkner, … , Frauke Zipp, Stefan Bittner
Katharina Birkner, … , Frauke Zipp, Stefan Bittner
Published October 29, 2019
Citation Information: J Clin Invest. 2020;130(2):715-732. https://doi.org/10.1172/JCI126381.
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Research Article Autoimmunity Neuroscience

β1-Integrin– and KV1.3 channel–dependent signaling stimulates glutamate release from Th17 cells

Abstract

Although the impact of Th17 cells on autoimmunity is undisputable, their pathogenic effector mechanism is still enigmatic. We discovered soluble N-ethylmaleimide–sensitive factor attachment receptor (SNARE) complex proteins in Th17 cells that enable a vesicular glutamate release pathway that induces local intracytoplasmic calcium release and subsequent damage in neurons. This pathway is glutamine dependent and triggered by binding of β1-integrin to vascular cell adhesion molecule 1 (VCAM-1) on neurons in the inflammatory context. Glutamate secretion could be blocked by inhibiting either glutaminase or KV1.3 channels, which are known to be linked to integrin expression and highly expressed on stimulated T cells. Although KV1.3 is not expressed in CNS tissue, intrathecal administration of a KV1.3 channel blocker or a glutaminase inhibitor ameliorated disability in experimental neuroinflammation. In humans, T cells from patients with multiple sclerosis secreted higher levels of glutamate, and cerebrospinal fluid glutamine levels were increased. Altogether, our findings demonstrate that β1-integrin– and KV1.3 channel–dependent signaling stimulates glutamate release from Th17 cells upon direct cell-cell contact between Th17 cells and neurons.

Authors

Katharina Birkner, Beatrice Wasser, Tobias Ruck, Carine Thalman, Dirk Luchtman, Katrin Pape, Samantha Schmaul, Lynn Bitar, Eva-Maria Krämer-Albers, Albrecht Stroh, Sven G. Meuth, Frauke Zipp, Stefan Bittner

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Figure 6

Immune-mediated neuronal Ca2+ elevations and neuronal damage can be significantly diminished by blockade of KV1.3 channels in vivo.

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Immune-mediated neuronal Ca2+ elevations and neuronal damage can be sign...
(A) EAE was induced in B6.Thy1-TN-XXLxRag2γc–/– mice (TN-XXL Rag–/–) via the passive transfer of CD4+ cells from B6.2D2.RFP.Th17 cells (Th17.RFP). At the peak of disease [d(max)], Ca2+ levels were imaged in vivo with intravital 2-photon imaging (TnC refers to troponin, a Ca2+ binding protein within the TN-XXL construct, comprising the genetically encoded calcium indicator). (B) Neuronal free Ca2+ levels were assessed by FRET measurements (false color-coded representation) before and after MgTX application (5 μM) in healthy control and EAE mice. Scale bars: 60 μm. B6.2D2.RFP.Th17 cells are shown in red. Ca2+ fluctuations were assessed by the 525 nm/475 nm ratio (ΔR/R). Sale bars: 30 μm. (C) Quantification of the full field 525 nm/475 nm ratio (ΔR/R) in healthy control mice (upper graphs, n = 32 control neurons, n = 48 MgTX neurons) and EAE mice (lower graphs, n = 33 control neurons, n = 42 MgTX neurons). Data were included from 7 control mice and 12 MgTX-treated mice (EAE score >2 from 2 independent experiments). (D) Changes in whole-field analysis of ratio channel intensities after application of 2 different KV1.3 blockers in vivo: MgTX (n = 7) and Shk-186 (n = 5). (E) Neuronal cortical cultures were incubated with Th17 cells for 12 hours with or without MgTX. Neurons were stained with Tuj1 and DAPI. Scale bars: 25 μm. (F) Quantification of viable neurons after application of Th17 cells with or without MgTX (n = 7–10) and T cell supernatant for 12 hours (n = 7). The y axis represents viable counts per 165 × 165 μm. Data represent the mean ± SEM. *P < 0.05, by unpaired Student’s t test (C) or 1-way ANOVA with Tukey’s post hoc test (D and F).