Airway inflammation and fear: elucidating immune mediators and neural substrates

PROJECT SUMMARY: Fear regulation is essential for optimal mental health. Maladaptive fear is a hallmark of posttraumatic stress disorder (PTSD), a debilitating condition afflicting 22% of combat veterans. Impaired functioning of infralimbic (IL) prefrontal cortex (PFC) contributes to fear dysregulation in PTSD, however factors contributing to IL deficits are unclear. Not all trauma-exposed individuals develop PTSD, suggesting that predisposition factors may contribute to IL dysfunction and PTSD risk. Elucidating the nature of such factors will help identify novel therapeutics. Growing evidence supports a strong association between severe asthma and PTSD. Mechanisms and cellular substrates whereby severe asthma associated factors regulate PTSD-relevant fear and IL-PFC deficits remain unknown. Using unique mouse paradigms of aeroallergen house dust mite (HDM)-driven inflammation we observe: 1) compromised fear extinction only in mice with allergen-induced Th2/Th17 expansion, an effect dependent on IL-17A and IL-17RA signaling and accompanied by reduced neuronal activation in the IL-PFC; 2) significant upregulation of microglial Il17ra expression in blood-brain-barrier compromised subfornical organ (SFO) inTh2/Th17 mice (but not in SFO non-microglial cells or PFC microglia); 3) IL-17A-induced activation of SFO neurons, and, 4) direct SFO►IL projections that modulate parvalbumin interneurons that regulate IL excitability and fear. Collectively these observations inform our hypothesis: IL-17A engages microglial IL-17RA and SFO-to-IL projections to modulate IL excitability and fear. This hypothesis will be tested in 2 aims. Aim 1 will determine if SFO►IL PFC projections regulate compromised fear extinction and IL neuronal excitability in mice with HDM-induced mixed Th2/Th17 inflammation. Using a retroCre- dependent chemogenetic strategy, we will inhibit or activate SFO►IL projections to assess effects on fear extinction in mice with Th2 versus Th2/Th17 responses. AAV-ChR2 transduction of SFO neurons and patch- clamp recordings in IL neurons will be undertaken. Aim 2 will determine if microglial IL-17RA signaling in the SFO drives HDM-induced fear extinction deficits. Using SFO targeted cell-specific AAV-Cre in Il17rafl/fl mice, we will assess the necessity of IL17RA signaling in HDM-induced extinction deficits. The transcriptional profile of SFO-derived microglia and non-microglial cells will be generated for cell-type specific gene expression signatures to identify DEGs and downstream signaling pathways in Th2/Th17 mice. Finally, the impact of IL-17A on IL-projecting SFO neurons will be assessed using patch-clamp electrophysiology in slices of brains Th2/Th17 mice, +/- targeted microglial IL-17RA ablation, inhibition of previously identified modulators of SFO microglia- neuron signaling and other transcriptomic-identified targets. Impact: Our studies will inform on how adaptive immune mediators modulate brain function and behavior and identify novel risk factors and therapeutic targets for fear-associated pathologies. Beyond asthma, our findings have implications for other conditions where IL- 17A is elevated in response to pulmonary pathologies (e.g. bacterial pneumonia, ARDS, COVID-19).