(420) Microtubule dysfunction as a mechanism of CF inflammation

Background: Previous data has identified distinct microtubule alterations in CF cells. By investigating the role of microtubule dysfunction in inflammatory regulation, potential therapeutic targets in Cystic Fibrosis (CF) can be identified. Previous work has shown that targeting microtubule regulation with HDAC6 inhibition corrects inflammatory signaling in both cell and mouse models of CF. Whether HDAC6 inhibition is acting through microtubules still needs to be determined. In this project we look at mouse nasal epithelial cells and how their inflammatory profile is changed when Tubulin polymerization-promoting protein/p25 (TPPP/p25), a protein known to directly affect microtubule structure and function, is knocked out. The mechanism through which this inflammation is mediated is interrogated, including assessment of inflammasome activation through NLRP3 oligomerization, and ultimately defining the mechanism of HDAC inhibition efficacy. Understanding the role that microtubule instability plays in inflammatory signaling creates an opportunity for therapeutic intervention in patients not eligible for CFTR correctors and/or patients with CF gene modifiers.

Methods: WT MNE, T3P KO MNE, and CF MNE cells were obtained through the CWRU CF Center cell culture core. Cells were exposed to IT to induce inflammation for 24 hours or no exposure for the control groups. Additional treatment groups included +/- HDAC6 inhibitor or vehicle (DMSO) for 48 hours prior to and during IT exposure. Following 24 hours of IT exposure, RNA samples were isolated. RNA samples were used to synthesize cDNA with the Quanta qScript cDNA synthesis kit. cDNA was used for quantitative RT-PCR using the TaqMan Gene expression assay. Actin cDNA was compared with other targets for delta delta CT quantification. Primers investigated include CXCL2 and IL-6. In vivo inflammatory responses were assessed using a mouse infection model. NLRP3 expression and cellular distribution were determined by Western blot and immunofluorescent imaging.

Results: After 24 hour exposure to IT, RNA fold difference for WT MNE cells was 4.99±2.27 for CXCL2 compared with 11.40±6.42 for T3P KO MNE cells (P< 0.05). Similarly, RNA fold difference for WT MNE cells was 1.69±0.41 for IL-6 compared with 3.41±1.57 for T3P KO MNE cells (P< 0.05). Preliminary results indicate improvement in inflammatory response following pretreatment with HDAC6 inhibition for 48 hours prior to inflammatory challenge with IT. Preliminary animal studies suggest impaired bacterial clearance and increased inflammatory cell recruitment compared to WT mice 3-days post-infection in Tppp/p25 KO mice, mimicking CF responses. There is evidence of increased oligomerization of NLRP3 in T3P cells compared with wild type cells at baseline as assessed via immunostaining suggesting a microtubule-related mechanism of increased inflammation.

Conclusions: TPPP/p25 KO MNE cells have a heightened inflammatory signaling response when compared with wild type as evidenced by increased production of CXCL2 and IL-6 mRNA production over WT MNE cells when challenged. TPPP/p25 KO mice also exhibit CF-like inflammation and reduced rates of bacterial clearance using an in vivo infection model. Cellular staining indicates that this inflammatory response is mediated through the inflammasome as evidenced by increased oligomerization of NLRP3. These data demonstrate that microtubule dysregulation is likely a key aspect of CF inflammatory responses and may offer insight into inflammatory mechanisms in CF.

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