Alpha-1-Antitrypsin Enhances Primary Human Macrophage Immunity Against Non-tuberculous Mycobacteria

Abstract

Rationale: The association between non-tuberculous mycobacterial lung disease and alpha-1-antitrypsin (AAT) deficiency is likely due, in part, to underlying emphysema or bronchiectasis. But there is increasing evidence that AAT itself enhances host immunity against microbial pathogens and thus deficiency could compromise host protection. Objectives: The goal of this project is to determine if AAT could augment macrophage activity against non-tuberculous mycobacteria. Methods: We compared the ability of monocyte-derived macrophages cultured in autologous plasma that were obtained immediately before and soon after AAT infusion-given to individuals with AAT deficiency-to control an ex vivo Mycobacterium intracellulare infection. Measurements and Main Results: We found that compared to pre-AAT infused monocyte-derived macrophages plus plasma, macrophages, and contemporaneous plasma obtained after a session of AAT infusion were significantly better able to control M. intracellulare infection; the reduced bacterial burden was linked with greater phagosome-lysosome fusion and increased autophagosome formation/maturation, the latter due to AAT inhibition of both M. intracellulare-induced nuclear factor-kappa B activation and A20 expression. While there was a modest increase in apoptosis in the M. intracellulare-infected post-AAT infused macrophages and plasma, inhibiting caspase-3 in THP-1 cells, monocyte-derived macrophages, and alveolar macrophages unexpectedly reduced the M. intracellulare burden, indicating that apoptosis impairs macrophage control of M. intracellulare and that the host protective effects of AAT occurred despite inducing apoptosis. Conclusion: AAT augments macrophage control of M. intracellulare infection through enhancing phagosome-lysosome fusion and autophagy.

Keywords: autophagy; mycobacteria; nuclear factor-kappa B; phagosome-lysosome fusion; serine protease inhibitor.

Figure 1

Effects of AAT infusion on macrophage control of M. intracellulare infection. (A) Mean plasma AAT levels ± SD, measured by nephelometry, pre- and post-AAT infusion of 10 PiZZ subjects. The normal range for plasma AAT at National Jewish Health is 0.72–1.92 mg/mL. (B) Quantitation of M. intracellulare in MDM cultured in plasma, both obtained before and after AAT infusion from PiZZ patients. Data shown are the mean ± SEM of cells from 10 subjects. (C) Pre-AAT infused MDM + post-AAT infused plasma (10 or 50%) vs. post-AAT infused MDM + pre-AAT infused plasma (10 or 50%) were infected with M. intracellulare. One hour, 2 and 4 days after infection, the cells were washed, lysed, and M. intracellulare quantified. Data shown are the mean ± SEM of cells from five subjects. (D) AAT (1, 2, 3, or 5 mg/mL) was incubated with a liquid culture of 5 × 10⁵ M. intracellulare/mL without macrophages for 2 and 4 days and M. intracellulare quantified. Data shown are the mean ± SD of three independent experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, NS, non-significant; Pre M+P, pre-AAT infused MDM + plasma; Post M+P, post-AAT infused MDM + plasma; AAT, alpha-1-antitrypsin; M. intra, M. intracellulare.

Figure 2

Quantitation of phagosome-lysosome fusion in MDM infected with M. intracellulare. (A) Monocytes obtained from PiZZ subjects pre- and post-AAT infusion were differentiated into macrophages and incubated with contemporaneous 10% and 50% plasma, and infected with GFP-M. intracellulare for 6 h. The cells were then stained for lysosomes with LysoTracker Red®, fixed, and stained with ProLong Gold Antifade Reagent with DAPI and placed in the dark at 4°C until analysis by fluorescent microscopy. Controls included MDM from healthy individuals incubated with 10% FBS, infected with M. intracellulare–GFP ± 10 U/mL IFNγ for 6 h. (B) The percentage of MDM with co-localization of M. intracellulare–GFP and lysosomes was calculated based on the number of cells with co-localization divided by the number of GFP-M. intracellulare infected cells. Data represent the mean ± SEM of cells from five subjects performed in duplicates. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, NS, non-significant.

Figure 3

Quantitation of autophagy in MDM infected with M. intracellulare. (A) MDM plus 10 or 50% plasma obtained pre- and post-AAT infusion were infected with M. intracellulare–GFP for 18 h, fixed, and immunostained for LC3B. The number of LC3B-positive intracellular particles was quantified by averaging the number of such particles per cell with 200 cells counted per condition for each experiment. Data represent the mean ± SEM of cells from six subjects performed in duplicates. (B) MDM plus 10 or 50% plasma obtained pre- and post-AAT infusion were infected with GFP-labeled M. intracellulare for 1 h, washed, and incubated for an additional three, 6 and 18 h. The cells were then fixed and immunostained for p62 and nuclei. The number of p62-positive intracellular particles was quantified by counting the number of p62-Cy3 positive particles per cell with 200 cells counted per condition for each experiment. Data represent the mean ± SEM of cells from three subjects performed in duplicates. (C) Quantitation of p65 subunit of NFκB binding to its consensus oligonucleotide in THP-1 cells with M. intracellulare infection alone or with 3 mg/mL AAT or 5 μM of the IκBα kinase inhibitor BAY 11-7082 (BAY) for 6 h. Data represent the mean ± SEM of three independent experiments performed in duplicates. (D) Quantitation of p65-NFκB binding in pre- and post-AAT infused MDM incubated in 10% contemporaneous plasma and infected with M. intracellulare alone or with AAT (3 mg/mL), BAY 11-7082 (5 μM), or both for 6 h. Data represent the mean ± SEM of cells from three subjects performed in duplicates. (E) Western blot of A20 protein expression in THP-1 cells with M. intracellulare infection alone or with AAT for 24 h. Immunoblot shown is representative of three independent experiments. A20 expression was semi-quantified, reported as the mean densitometry from three independent experiments. Pre M+P, pre-AAT infused MDM + plasma; Post M+P, post-AAT infused MDM + plasma; M. intra, M. intracellulare. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, NS, non-significant.

Figure 4

AAT-induced autophagy enhances macrophage control of M. intracellulare. (A) Pre- and post-AAT infused MDM and contemporaneous 10 or 50% plasma were infected with M. intracellulare for 24 h, nuclear-free cell lysate separated by SDS-PAGE, and immunblotted for A20. Data represent the mean ± SEM of cells from three subjects in duplicates. (B) Pre- and post-AAT infused MDM and plasma were infected with M. intracellulare for 1, 2, and 4 days and in a separate aliquot of post-AAT infused MDM + plasma, 8 mM 3-methyladenine (3-MA) was added simultaneously with the infection. Data represent the mean ± SEM of cells from three subjects in duplicates. ^*p < 0.05, ^**p < 0.01; Pre M+P, pre-AAT infused MDM + plasma; Post M+P, post-AAT infused MDM + plasma; M. intra, M. intracellulare.

Figure 5

Quantitation of cytokines in the cell culture supernatants. At the indicated conditions and times of infection, the cell culture supernatant of the MDM were assayed for (A) IFNγ, (B) IL-1β, (C) IL-6, (D) TNFα, (E) IL-10, (F) IL-8, and (G) IL-12 (p70). Data shown are the mean ± SD of cells from 7 to 8 subjects performed in duplicates. ^*p < 0.05, ^**p < 0.01, NS, non-significant.

Figure 6

AAT induced apoptosis of macrophages infected with M. intracellulare. (A) MDM plus 10 or 50% plasma obtained pre- and post-AAT infusion were infected with M. intracellulare, incubated for two days with either 10% FBS or 10 or 50% pre- and post-AAT infusion plasma from PiZZ subjects, and TUNEL assay performed. Data shown are the mean ± SEM of cells from eight subjects performed in duplicates. (B) Differentiated THP-1 cells were infected with M. intracellulare alone or with AAT or with both AAT and z-DEVD-fmk for 24 h, lysed, and activated caspase-3 quantified by ELISA. Data represent the mean ± SEM of three independent experiments performed in duplicates. (C) Caspase-3 activity was measured in pre-AAT infused MDM in unstimulated cells or cells infected with M. intracellulare alone or with AAT ± z-DEVD-fmk for 24 h. Data represent the mean ± SEM of cells from three subjects in duplicates. (D) Liquid cultures of M. intracellulare at 5 × 10⁵ mycobacteria per mL were incubated with 0.1% DMSO or 1, 3, 5, and 10 μM z-DEVD-fmk for 2 and 4 days, followed by quantitation of viable M. intracellulare. Data represent the mean ± SEM of three independent experiments performed in duplicates. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, NS, non-significant; Pre M+P, pre-AAT infused MDM + plasma; Post M+P, post-AAT infused MDM + plasma; M. intra, M. intracellulare.

Figure 7

Inhibition of caspase-3 reduced M. intracellulare burden in macrophages. (A) M. intracellulare growth in THP-1 cells ± AAT or caspase 3 inhibitor z-DEVD-fmk (10 μM) at 1 h, 2, and 4 days after infection. Data shown are the mean ± SEM of five independent experiments performed in duplicates. (B) MDM derived from pre-AAT infused monocytes from PiZZ patients incubated with 10% FBS were infected with M. intracellulare alone or with AAT (3 or 5 mg/mL), 10 μM z-DEVD-fmk, and both 5 mg/mL AAT and 10 μM z-DEVD-fmk. After the indicated times, cell-associated M. intracellulare were quantified. Data shown are the mean ± SEM of cells from four subjects performed in duplicates. (C) MDM derived from post-AAT infused monocytes and plasma incubated with 3 mg/mL exogenous AAT or 10 μM z-DEVD-fmk and infected with M. intracellulare for the indicated times and cell-associated M. intracellulare quantified. Data shown are the mean ± SEM of cells from three subjects performed in duplicates. (D) Primary human alveolar macrophages from organ donors were cultured as described in Experimental Methods and incubated with AAT or z-DEVD-fmk at the indicated concentrations for 1 h, and 2 and 4 days, followed by quantitation of M. intracellulare. Data shown are the mean ± SEM of cells from six cadaveric donors performed in duplicates. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 8

Cartoon of the mechanism by which AAT enhances macrophage control of M. intracellulare infection. The paradigm from the results of this study is that AAT inhibits p65-NFκB activation in M. intrcellulare-infected macrophages, resulting in inhibition of autophagy and increased susceptibility to M. intracellulare infection in macrophages. It is also likely that AAT is inducing other host-protective mechanisms in macrophages that are independent of p65-NFκB activation. Based on the work of others, increased autophagy may further decrease A20, further amplifying autophagy and providing a positive feedback loop (dashed blue arrow); however, decreased A20 expression may attenuate AAT inhibition of NFκB, preventing excessive inhibition of NFκB by AAT and displaying a negative feedback loop (dashed inhibitory red arrow).