Lester, S. E. | bioRxiv Science

bioRxiv2024

Autophagy is suppressed in peripheral blood mononuclear cells during chronic obstructive pulmonary disease.

Assessing autophagy may offer insights into the pathogenesis of chronic obstructive pulmonary disease (COPD). However, measuring the dynamic aspect of autophagy is challenging, and sample manipulation can cause signal fluctuations that deviate from physiological conditions. We applied an organotypic method to quantify autophagy in COPD, where it frequently demonstrates disease-related dysregulation. Blood from control and COPD participants were treated with or without chloroquine. LC3B-II abundance was quantified in peripheral blood mononuclear cells, and findings were validated by transmission electron microscopy. Our observations show that while basal LC3B-II abundance was similar between groups (P = 0.60), autophagic flux was significantly lower in the COPD cohort, suggesting disruption in the regulatory factors that direct autophagosome clearance (P = 0.004). This was supported by less frequent observations of autophagy-related vacuoles in the cytosol of COPD-derived PBMCs. Our findings indicate that the suppression of autophagy can be detected in the blood of individuals with COPD, which warrants further investigation into its contribution to extrapulmonary disease processes.

molecular biology↗

bioRxiv2024

Modification of Azithromycin to Mitigate its Arrest of Autophagy

Aims/hypothesisAutophagy plays a critical role in the survival and microbial clearance function of tissues that encounter the environment, such as airway epithelial cells (AECs). Contrary to the known roles of azithromycin (AZM) in promoting microbial clearance, our evidence shows that AZM is a potent inhibitor of autophagy - an effect consistent with bacterial residency. Here we investigate the structure-activity relationship of AZM vs other macrolides and AZM-3-N-oxide (AZM-[O]), to mitigate the off-target arrest of autophagy. MethodAZM-[O] was synthesised in-house via selective oxidation of the desosamine amine of AZM. Human peripheral blood mononuclear cells (PBMC) were used to assess autophagy ex vivo in an organotypic manner via transmission electron microscopy and Western blot analysis. For in vitro studies, the 16HBE14o-AEC line and Western blot was used to assess macrolide vs autophagy structure-activity relationships, and autophagic flux by quantifying the protein abundance of LC3B-II vs Sequestosome-1. Subsequent assessments of antimicrobial activity were conducted using the micro-broth dilution method. Immunomodulatory outcomes were assessed by quantifying the secretion of IL-6 in a lipopolysaccharide PMA-stimulated THP-1 macrophage model. ResultsAZM significantly inhibited autophagic flux in both the ex vivo PBMC and in vitro 16HBE14o-AEC models, evidenced by the accumulation of autophagosome-related vacuoles and LC3B-II and Sequestosome-1 protein, compared to its precursors and other macrolides including roxithromycin and clarithromycin. Notably, oxidation of AZM to produce AZM-[O] significantly alleviated this inhibitory effect on autophagy, but without completely preserving its antimicrobial and immunomodulatory functions. Conclusion/DirectionsWe show for the first time that AZM inhibits PBMC autophagy in human blood ex vivo and there is a high probability this phenomenon occurs in the clinical setting. Importantly, chemical modification of AZM to generate AZM-[O] substantially alleviated this effect, consistent with altered ionisation properties. We are now assessing clinically derived blood samples in participants treated with AZM, defining the AZM-mammalian protein interactome and developing further AZM derivatives that preserve immunomodulatory activity while minimising disruption of autophagy.

biochemistry↗