Anti-inflammatory IL-4 shapes the architecture of presynaptic glutamatergic puncta of human and murine sensory neurons for pain sensitivity

The pleiotropic anti-inflammatory cytokine interleukin 4 (IL-4) not only modulates immune responses but also plays important roles in the nervous system by mediating itch, antinociception and neuroregeneration. Here, we assessed the expression and function of IL-4, its receptors, and downstream signaling pathways in human induced pluripotent stem cell derived nociceptors (iNocs), organotypic mouse spinal cord cultures, and in in vivo models. In silico analyses of human and mouse dorsal root ganglion (DRG) single cell transcriptomes revealed that a specific subpopulation of murine and human sensory neurons expressed IL4R and IL13RA1 (but very little IL2RG) receptor subunits highlighting conserved neuron responsiveness to IL-4. In iNocs, exogenous IL-4 induced de novo formation of presynaptic vGLUT1-positive puncta. Pharmacological inhibition of Janus kinases 1/2, STAT6, protein kinase C isoforms, or the translation initiation factor eIF4E significantly attenuated the IL-4-associated increase in vGLUT1-positive puncta. Transcriptomic profiling of iNocs revealed IL-4-regulated biological processes associated with neuronal development and synaptic organization. Consistently, IL-4 increased mean-firing-rate in iNocs and increased vGLUT1 expression in mouse organotypic spinal cord cultures treated with IL-4, in vitro. Intrathecal or intranasal IL-4 treatment enhanced both, inhibitory and excitatory synaptic activity in acute spinal cord slices, and this was associated with a decrease in mechanical and thermal sensitivity but normal sensorimotor coordination in vivo. These findings position IL-4 as key regulator of presynaptic glutamatergic organization for primary afferent neuron connectivity in the spinal dorsal horn and support a critical role for IL-4 in the excitation - inhibition balance of pain processing spinal neuronal networks.