Preterm ovine respiratory epithelial cell responses to mechanical ventilation, lipopolysaccharide, and interleukin-13.

Mechanical ventilation causes airway injury, respiratory epithelial cell proliferation, and lung inflammation in preterm sheep. Whether preterm epithelial cells respond similarly to adult epithelial cells or are altered by mechanical ventilation is unknown. We test the hypothesis that mechanical ventilation alters the responses of preterm airway epithelium to stimulation in culture. Respiratory epithelial cells from the trachea, left mainstem bronchi (LMSB), and distal bronchioles were harvested from unventilated preterm lambs, ventilated preterm lambs, and adult ewes. Epithelial cells were grown in culture or on air-liquid interface (ALI) and challenged with combinations of either media only, lipopolysaccharide (LPS; 10 ng/mL), bronchoalveolar fluid (BALF), or interleukin-13 (IL-13). Cell lysates were evaluated for mRNA changes in cytokine, cell type markers, Notch pathway, and acute phase markers. Mechanical ventilation altered preterm respiratory epithelium cell types. Preterm respiratory epithelial cells responded to LPS in culture with larger IL-8 induction than adults, and mechanical ventilation further increased cytokines IL-1ß and IL-8 mRNA induction at 2 h. IL-8 protein is detected in cell media after LPS stimulation. The addition of BALF from ventilated preterm animals increased IL-1ß mRNA to LPS (fivefold) in both preterm and adult cells and suppressed IL-8 mRNA (twofold) in adults. Preterm respiratory epithelial cells, when grown on ALI, responded to IL-13 with an increase in goblet cell mRNA. Preterm respiratory epithelial cells responded to LPS and IL-13 with responses similar to adults. Mechanical ventilation or exposure to BALF from mechanically ventilated animals alters the responses to LPS.NEW & NOTEWORTHY Preterm lamb respiratory epithelial cells can be extracted from the trachea and bronchi and frozen, and the preterm cells can respond in culture to stimulation with LPS or IL-13. Brief mechanical ventilation changes the distribution and cell type of preterm respiratory cells toward an adult phenotype, and mechanical ventilation alters the response to LPS in culture. Bronchoalveolar lavage fluid from preterm lambs receiving mechanical ventilation also alters unventilated preterm and adult responses to LPS.

A model of toxic neuropathy in Drosophila reveals a role for MORN4 in promoting axonal degeneration.

Axonal degeneration is a molecular self-destruction cascade initiated following traumatic, toxic, and metabolic insults. Its mechanism underlies a number of disorders including hereditary and diabetic neuropathies and the neurotoxic side effects of chemotherapy drugs. Molecules that promote axonal degeneration could represent potential targets for therapy. To identify such molecules, we designed a screening platform based on intoxication of Drosophila larvae with paclitaxel (taxol), a chemotherapeutic agent that causes neuropathy in cancer patients. In Drosophila, taxol treatment causes swelling, fragmentation, and loss of axons in larval peripheral nerves. This axonal loss is not due to apoptosis of neurons. Taxol-induced axonal degeneration in Drosophila shares molecular execution mechanisms with vertebrates, including inhibition by both NMNAT (nicotinamide mononucleotide adenylyltransferase) expression and loss of wallenda/DLK (dual leucine zipper kinase). In a pilot RNAi-based screen we found that knockdown of retinophilin (rtp), which encodes a MORN (membrane occupation and recognition nexus) repeat-containing protein, protects axons from degeneration in the presence of taxol. Loss-of-function mutants of rtp replicate this axonal protection. Knockdown of rtp also delays axonal degeneration in severed olfactory axons. We demonstrate that the mouse ortholog of rtp, MORN4, promotes axonal degeneration in mouse sensory axons following axotomy, illustrating conservation of function. Hence, this new model can identify evolutionarily conserved genes that promote axonal degeneration, and so could identify candidate therapeutic targets for a wide-range of axonopathies.