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A4308 - Alpha-1-Antitrypsin Enhances Macrophage Control of Mycobacterium Intracellulare Infection Despite Inducing Macrophage Apoptosis
Author Block: E. D. Chan, A. Bai, J. R. Honda, A. Musheyev, Z. F. Feng, G. Huitt, R. Harbeck, R. A. Sandhaus, X. Bai; Natl Jewish Med Ctr, Denver, CO, United States.
RATIONALE: The occurrence of non-tuberculous mycobacterial lung disease (NTM-LD) is increased in individuals with alpha-1-antitrypsin (AAT) deficiency. We previously showed that monocyte-derived macrophages (MDM) and temporaneous plasma of PiZZ subjects obtained after AAT infusion were better able to control an ex vivo Mycobacterium intracellulare infection than MDM and plasma obtained immediately prior to an AAT infusion. Additionally, we have reported that post-AAT infused MDM and plasma infected with M. intracellulare have increased phagosome-lysosome fusion, autophagosome formation and maturation, and increased expression of pro-inflammatory cytokines and a trend toward reduced interleukin-10 production. The goal of this current project is to determine the effect of AAT augmentation on apoptosis and nuclear factor-kappa B (NF-kappaB) activation of M. intracellulare-infected macrophages. METHODS: Peripheral blood mononuclear cells (PBMC) were differentiated into MDM with M-CSF, infected the MDM with M. intracellulare in the presence of pre- or post-AAT infused plasma, and TUNEL assay performed. RESULTS: We found that MDM incubated with post-AAT-infused plasma and infected with M. intracellulare had a modest but significant increase in apoptosis compared to MDM incubated with pre-AAT infused plasma. Moreover, AAT enhanced caspase-3 activity in differentiated THP-1 macrophages infected with M. intracellulare compared with infected cells without AAT. Pre-AAT infused MDM incubated with exogenous AAT (3 or 5 mg/mL) also reduced the intracellular burden of M. intracellulare. However, pharmacologic inhibition of caspase-3 in pre-AAT infused MDM infected with M. intracellulare significantly reduced the intracellular burden of bacteria, indicating that classical apoptosis impairs macrophage control of M. intracellulare infection and that AAT reduced macrophage burden of M. intracellulare despite its induction of apoptosis. Since nuclear factor-kappa B (NF-kappaB) is often considered to be anti-apoptotic, we determined the effect of exogenous AAT (3 mg/mL) on NF-kappa B activation with M. intracellulare infection of THP-1 macrophages and found that M-AAT significantly decreased p65 subunit of NFkB binding to its canonical cis-regulatory element, consistent with a previous known effect of normal M-AAT. Immunoblotting showed that AAT increased cytochrome C expression in a dose-dependent fashion in THP-1 macrophages infected with M. intracellulare. CONCLUSION: We conclude from these findings that - unlike the anti-apoptotic effects of AAT on endothelial and epithelial cells - AAT increases apoptosis of M. intracellulare-infected macrophages. Despite the finding that apoptosis of macrophages is host deleterious with M. intracellulare infection, AAT enhanced macrophage control of M. intracellulare.