Background & Aims

The anti-inflammatory type 2 cytokine interleukin-4 (IL-4) is generated and secreted by a variety of immune cells including microglia or T-cells. IL-4 not only acts back on immune cells (1) but also has important roles in the nervous system mediating antinociception and neuroregeneration (2). In the central nervous system, IL-4 improves neuronal outgrowth and repair through pathways involving presynaptic vesicular glutamate transporter (vGLut1) (3). In the peripheral nervous system, upon nerve injury, IL-4 promotes nerve regeneration in rodents, and IL-4 depletion compromises regenerative processes (4). To study the effect of IL-4 on its receptors and downstream signaling pathways in human nociceptors, we assessed expression of IL-4R and IL-13RA subunits in differentiating human induced pluripotent stem cell (iPSC) derived nociceptors (iNocs) and investigated IL-4 induced morphological, transcriptional and electrophysiological alterations.

Methods

iNocs were differentiated from human iPSC (clone STBCi026B) from pluripotency to neuronal progenitors before maturation until day 16 (5) where Il-4 receptor forming subunits IL4R and IL13RA1 were identified and validated using the Nociceptra app (6). IL-4 (20 ng/ml) was applied for 24 h at day 15. A pharmacological approach was used to assess signalling components and for this, cells were pretreated with STAT 6 inhibitor AS1517499 (1µM), eIF4E/eIF4G interaction inhibitor 4EGI-1 (40 µM), mammalian target of rapamycin (mTOR) inhibitor rapamycin (200nM) and protein kinase C (PKC) inhibitor bisindolylmaleimide 1 (1µM). Cells were harvested at d16 for RNA sequencing, immunofluorescence microscopy and microfluorimetric Ca2+ measurements.

Results

iNocs expressed both components of the IL-4 receptor complex (IL-4ra and IL-13ra1) and signaling machinery (Jak1,2, STAT5, PKC isoforms, translation factor EiF4E) during differentiation. IL-4 induced de novo formation of boutons immunoreactive for vGLUT1 after 24 h of treatment which was not observed upon pharmacological inhibition of translational and cellular signaling components. RNA-seq unveiled distinct transcriptomic changes characterized by 932 significantly up- and 1577 downregulated genes in Il-4 treated iNocs. GO-analysis revealed biological process ontologies for “neuron projection development”, “axonogenesis” and “synapse”, “cellular process involved in reproduction in multicellular organism”, “regulation of membrane potential” and “calcium ion transmembrane transport”. Comparison with publicly available peripheral nerve injury data sets in mice revealed 9 commonly downregulated and 46 upregulated genes after peripheral nerve injury in mice and IL-4 treatment in iNocs.

Conclusions

IL-4 led to massive changes in transcript levels of proteins that are essential for the structure of glutamatergic synapses. These alterations partially reflect injury-induced transcriptional changes from mouse nerve injury models contributing to regenerative processes in peripheral nerve injury but possibly also to reconnecting primary afferent neurons to their projections in the spinal dorsal horn indicative of a more general role of IL-4 in the control of developing neuronal networks.

References

1. Gieseck, R. L., 3rd, Wilson, M. S., & Wynn, T. A. (2018). Type 2 immunity in tissue repair and fibrosis. Nat Rev Immunol, 18(1), 62-76. doi:10.1038/nri.2017.90
2. Kiyota, T., Okuyama, S., Swan, R. J., Jacobsen, M. T., Gendelman, H. E., & Ikezu, T. (2010). CNS expression of anti-inflammatory cytokine interleukin-4 attenuates Alzheimer’s disease-like pathogenesis in APP+PS1 bigenic mice. Faseb j, 24(8), 3093-3102. doi:10.1096/fj.10-155317
3. Hanuscheck, N., Schnatz, A., Thalman, C., Lerch, S., Gärtner, Y., Domingues, M., . . . Vogelaar, C. F. (2020). Growth-Promoting Treatment Screening for Corticospinal Neurons in Mouse and Man. Cell Mol Neurobiol, 40(8), 1327-1338. doi:10.1007/s10571-020-00820-7
4. Walsh, J. T., Hendrix, S., Boato, F., Smirnov, I., Zheng, J., Lukens, J. R., . . . Kipnis, J. (2015). MHCIIindependent CD4+ T cells protect injured CNS neurons via IL-4. J Clin Invest, 125(2), 699-714. doi:10.1172/jci76210
5. Schoepf, C. L., Zeidler, M., Spiecker, L., Kern, G., Lechner, J., Kummer, K. K., & Kress, M. (2020). Selected Ionotropic Receptors and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (iPSC)-Derived Nociceptors. Brain Sciences, 10(6). https://doi.org/10.3390/BRAINSCI10060344
6. Zeidler, M., Kummer, K. K., Schöpf, C. L., Kalpachidou, T., Kern, G., Cader, M. Z., & Kress, M. (2021). NOCICEPTRA: Gene and microRNA Signatures and Their Trajectories Characterizing Human iPSC?Derived Nociceptor Maturation. Advanced Science, 8(21). https://doi.org/10.1002/ADVS.202102354

Presenting Author

David Zimmermann

Poster Authors

David Zimmermann

MSc

Medical University Innsbruck

Lead Author

Clemens Lukas Schöpf

PhD

Lead Author

Georg Kern

Lead Author

Theodora Kalpachidou

Medical University of Innsbruck

Lead Author

Maximilian Zeidler

Lead Author

Michaela Kress

Institute of Physiology, Medical University of Innsbruck, Innsbruck, Austria

Lead Author

Topics

  • Mechanisms: Biological-Molecular and Cell Biology