B33-22 The Role of Airway Epithelial LMAN1 in Activation of the Inflammasome in Response to Alternaria Alternata

Rationale

Our lab has previously identified LMAN1 as a receptor for house dust mite allergens and recent data indicates that LMAN1 can also bind allergens from Alternaria alternata (A. alternata), a fungus associated with asthma exacerbation and severity. Global loss of LMAN1 resulted in sex-dependent effects on airway hyperresponsiveness and immune cell recruitment in an A. alternata asthma model. In order to determine molecular mechanisms for such results, untreated or A. alternata- treated WT and LMAN1 KO BEAS-2B cells were subjected to RNAseq and inflammatory cytokine multiplex assays. RNAseq implicated LMAN1 in processes related to mitochondrial function, cell death/survival, and the immune response; while multiplex assays showed LMAN1-dependent secretion of IL-1β. These data led us to examine whether LMAN1 plays a role in activation of the inflammasome in airway epithelial cells (AECs) in response to A. alternata.

Methods

In order to determine involvement of LMAN1 in inflammasome activation, we utilized WT and LMAN1 KO BEAS-2B cells and left them untreated, or treated them with A. alternata. Inflammasome regulated cytokine secretion (IL-1β and IL-18) was assessed by ELISA. Various inducers of inflammasome activation, such as ROS, Ca2+, and mtDNA release, were measured using MitoSOX, Fluo-4AM, and cellular fractionation, respectively. Mitochondrial health and function were assessed using MMP and Seahorse assays. Effects on A. alternata -induced cell death were measured using Annexin/PI, caspase activation, and LDH assays. Lastly, levels and activation states of inflammasome components were assessed using Western Blot.

Results

Loss of LMAN1 in airway epithelial cells resulted in reduced IL-1β but not IL-18 cytokine secretion, diminished A. alternata -induced ROS and Ca2+ production, improved acute mitochondrial fitness and mitochondrial respiration, and improved cell survival.

Conclusion

Overall, our current data suggests that LMAN1 within AECs fulfills multiple roles - contributing to A. alternata-induced inflammasome activation, but at the same time, improving mitochondrial and cellular fitness. These findings provide important considerations if pursuing cell-specific targeting of LMAN1 in allergic airway disease.

This abstract is funded by: NIH R01HL162897