Transient Interleukin 6 elevation at early developmental stages deregulates glutamatergic synaptogenesis and brain connectivity

Prenatal development of the brain follows a precise program that ensures establishing a proper neuronal network in adulthood. Brain development is influenced by several molecules, including cytokines. An increase in the amount of cytokines due to an inflammatory challenge during pregnancy, called Maternal Immune Activation (MIA), can lead to deleterious effects on the development of the fetus’ brain. Indeed, MIA is associated with a higher susceptibility for the offspring to develop neurodevelopmental disorders (NDDs) such as Autism Spectrum Disorder and Schizophrenia. A common feature of NDDs is a synaptic disfunction and a subsequent network alteration supported by an unbalance of Excitatory/Inhibitory (E/I) ratio. The pro-inflammatory cytokine interleukin 6 (IL-6) was found to be central in MIA, although its precise role in neuronal and synaptic development is unknown. The aim of this work is to understand the molecular mechanisms by which IL-6 impacts on the formation of the brain and synapses. A single injection of IL-6 in pregnant mice at gestational day 15 induced an increase of excitatory synapse density in pyramidal hippocampal neurons of the offspring that is maintained in the adulthood. The net effect of IL-6 is a glutamatergic pro-synaptogenic boost yielding to an increased E/I ratio. Furthermore, the increased hippocampal synaptic density sustains a functional hyperconnection of hippocampal networks detected in the offspring of IL-6-MIA model by scanning the resting state functional Magnetic Resonance Imaging. The augmented excitatory transmission could be recapitulated in primary hippocampal cultures by a single IL-6 treatment in the early phase of neuronal development, pointing out the consistency of IL-6 synaptic effect both in vivo and in vitro. We found the transcription factor STAT3 plays a critical role in IL-6 dependent increase of glutamatergic synapses, which can be prevented by the blockade of its genomic activity. IL-6 stimulation in hippocampal neurons triggers STAT3 activation by phosphorylation of the transcriptional factor. We showed that the constitutive activation of STAT3 signaling in cell autonomous manner is sufficient to induce the synaptic effect of IL-6. The specificity of IL-6 effect on neuronal function was further corroborated by the persistence of the pro-synaptogenic effect of IL-6 in absence of astrocytes component of the cultures. Using single-cell sequencing we identified an enrichment of Regulatory of G protein Signal (RGS) 4 in neuronal clusters upon IL-6 stimulation. In addition, we validate RGS4 as a downstream target gene of STAT3 and the fundamental activity of RGS4 in the IL-6 pro-synaptogenic effects. The upregulation of STAT3 and RGS4 were also validated in vivo, 24 hours after the IL-6 or Poly I:C (the canonical MIA model) stimulation. The involvement of RGS4 in the MIA model is a novel insight on the pathological mechanism of maternal immune stimulation, that further underscores the role of IL-6 in the MIA context. This work highlights the role of Interleukin 6 in brain development suggesting a trophic function of this cytokine in promoting glutamatergic synaptogenesis through RGS4-mediated signaling and proposes STAT3-RGS4 as a new pathway to better investigate in the effect of maternal immune stimulation.