https://www.atsjournals.org/doi/pdf/10.1164/ajrccm-conference.2024.209.1_MeetingAbstracts.A5028
Am J Respir Crit Care Med 2024;209:A5028
C20 HAVINGA GROWTH MINDSET: REPROGRAMMINGCELLULAR PATHWAYS TO DISEASE / Mini Symposium / Tuesday, May 21/09:15 AM-11:15 AM / San Diego Convention Center, Room 30A-B (Upper Level)
C. Koster, B. Q. Wang, S. Bos, M. H. de Jager, P. Mulder, E. M. J. Verpoorte, B. N. Melgert, R. Gosens
Rationale: Neuroplasticity is defined as the ability of the nervous system to reorganize its structure, function and connections. In asthma this manifests as increased neuron density and neurite length as well as a lowered firing threshold, resulting in hyperresponsiveness to (a-)specific stimuli. An increase in the number of mast cells in the direct vicinity of the sensory neurons in the airways of fatal asthma patients was recently observed. However, little is known on how sensory neurons regulate mast cell function. Therefore, we aimed to study the interaction of sensory neurons with mast cells. To achieve this, we developed an in vitro co-culture model of human sensory neurons and mast cells to study neuroimmune interactions. Methods: Using a 35-day differentiation protocol, H9WA09 human pluripotent stem cells (hPSCs) were differentiated into sensory neurons. After maturation of the hPSC-derived sensory neurons, a co-culture was established with mast cells (LUVA) in a 50/50-mixture of sensory neuron maturation and LUVA medium. After 5 days of co[1]culture, the two cell types were separated using PSA-NCAM microbeads followed by RNAseq analysis. Additionally, Ca 2+ -measurements were performed to assess the activity of the sensory neurons with and without exposure to mast cells. Results: The successful generation of β3- tubulin + /TRPV1 + sensory neurons, both in the presence and absence of mast cells (FcεRI + ) was confirmed using immunofluorescence and flow cytometry. Additionally, co-cultures were established in the axon-guiding NeuroChip. Co-culture did not affect β3-tubulin + /TRPV1 + sensory neuron differentiation. However, RNAseq analysis of the mast cell population showed enhanced mast cell differentiation in co-culture, as well as upregulation of 10/86 asthma susceptibility genes. GSEA analysis showed upregulation of multiple immune-related pathways. Including INFLAMMATORY_RESPONSE, as well as IL2_STAT5, IL6_JAK_STAT3, TGF_BETA and TNFA_SIGNALING_VIA_NFKB. Furthermore, the upregulation of several Th2-immune response[1]related genes was observed, such as IL6, ARG2, ALOX5 and LTA4H. Ca 2+ -measurements of the sensory neurons showed increased responsiveness of the mast cell exposed sensory neurons compared to monocultured sensory neurons, upon addition of the TRPV1 agonist capsaicin (10nM[1]10µM). Conclusions: We successfully established an in vitro co-culture model of hPSC-derived sensory neurons and mast (LUVA) cells, which facilitates further studies of neuro-effector interactions between the two cell types. We now show induction of mast cell differentiation and immune activation upon co-culture with the sensory neurons with increased sensitivity of the sensory neurons. These findings implicate that an increased presence of mast cells in asthmatic lungs lowers the firing threshold of sensory neurons and increased sensitivity of the airways.
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