Epithelial and macrophage dysfunction in alpha1-antitrypsin deficiency

Alpha-1-antitrypsin is produced and secreted from the liver. It plays an important role in controlling inflammation and preventing tissue damage, particularly in the lung. A common genetic mutation in the 1-antitrypsin gene causes molecules of 1-antitrypsin to stick together, forming polymers. Polymers are trapped in liver cells causing liver damage. The amount of 1-antitrypsin in the blood is reduced and cannot perform its usual function; this causes lung inflammation and emphysema. Alpha-1-antitrypsin is also produced in cells that line the lung and in white blood cells. I propose to assess whether the retention of polymers in these cells causes them to malfunction and so cause further lung damage. I will do this by expressing the mutant protein in cell lines and assessing whether this causes them to malfunction and release inflammatory molecules. I will then take samples from the lungs of individuals with 1-antitrypsin deficiency to see if the cells are dysfunctional in humans. This will allow me to assess whether the retention of polymers of mutant 1-antitrypsin in lung cells contributes to lung inflammation and hence emphysema in individuals with 1-antitrypsin deficiency.

1-antitrypsin is the most abundant circulating protease inhibitor. The common severe Z allele (Glu342Lys) causes the protein to form ordered polymers that are retained within the endoplasmic reticulum (ER) of hepatocytes. These polymers do not activate the unfolded protein response (UPR) but lead to activation of NF-kB. This in turn causes liver disease whilst the lack of circulating 1-antitrypsin predisposes the PiZZ homozygote to early onset emphysema. Z 1-antitrypsin is synthesised and secreted by monocytes/macrophages and bronchial and alveolar epithelial cells within the lung. It is unknown whether the accumulation of polymers within these cells also activates NF-kB as they do in the liver. I propose to evaluate this with 2 specific aims: (i) to determine the effect of intracellular polymers on epithelial cells in vitro and (ii) to determine the effect of intracellular polymers on lung epithelium and monocytes/macrophages in vivo.

I will firstly generate alveolar (A549) and bronchial epithelial cell lines (BEAS-2B) that conditionally express wildtype and Z 1-antitrypsin under the control of a Tet-ON promotor. I will then assess whether the expression of wildtype or Z 1-antitrypsin causes the activation of the unfolded protein response (by measuring BiP, Grp94, GADD34 and CHOP protein levels, XBP1 mRNA splicing and ATF6 reporter activation), ER calcium release (Fluo-4 fluorescence), NF- B signalling (NF- B subunit protein levels, subcellular localisation and reporter activation) and the release of inflammatory cytokines such as IL-6 and IL-8 by sandwich ELISA. In the second aim I will purify monocytes from the blood of individuals with PiZZ 1-antitrypsin deficiency and PiMM controls. I will assess whether the monocytes that express Z 1-antitrypsin show evidence of ER stress, calcium release and a greater release of inflammatory cytokines. Finally, I will bronchoscope and take bronchial biopsies from individuals with PiZZ 1-antitrypsin deficiency and PiMM controls to assess whether epithelial cells in vivo show similar changes to those see in vitro.

Taken together these experiments will allow me to determine whether the accumulation of Z 1-antitrypsin causes epithelial and monocyte/macrophage dysfunction in 1-antitrypsin deficiency. If so, then this would provide novel insights into the pathogenesis of the associated emphysema.