Autophagic Killing Effects against <i>Mycobacterium tuberculosis</i> by Alveolar Macrophages from Young and Aged Rhesus Macaques

<div><p>Non-human primates, notably rhesus macaques (<i>Macaca mulatta</i>, RM), provide a robust experimental model to investigate the immune response to and effective control of <i>Mycobacterium tuberculosis</i> infections. Changes in the function of immune cells and immunosenescence may contribute to the increased susceptibility of the elderly to tuberculosis. The goal of this study was to examine the impact of age on <i>M. tuberculosis</i> host-pathogen interactions following infection of primary alveolar macrophages derived from young and aged rhesus macaques. Of specific interest to us was whether the mycobactericidal capacity of autophagic macrophages was reduced in older animals since decreased autophagosome formation and autophagolysosomal fusion has been observed in other cells types of aged animals. Our data demonstrate that alveolar macrophages from old RM are as competent as those from young animals for autophagic clearance of <i>M. tuberculosis</i> infection and controlling mycobacterial replication. While our data do not reveal significant differences between alveolar macrophage responses to <i>M. tuberculosis</i> by young and old animals, these studies are the first to functionally characterize autophagic clearance of <i>M. tuberculosis</i> by alveolar macrophages from RM.</p></div

Comparison of innate immune functions of alveolar macrophages of adult and aged RM.

<p>A. Phagocytic capacity of alveolar macrophages from adult and aged RM was measured. Alveolar macrophages were incubated with fluorescently labeled <i>M. tuberculosis</i> CDC1551 for 45 minutes, and then extracellular bacteria removed by washing. Cells were fixed, and infected macrophages were observed by fluorescence microscopy. A macrophage was considered infected if it had internalized at least one bacterium. Each symbol represents the % infection of each RM sample. The average and standard deviation of all samples for each group is shown. n>100 macrophages for each RM sample. B. Alveolar macrophages from aged RM were infected at an MOI of 5∶1 with <i>M. tuberculosis</i> CDC1551. Bacterial cfu were determined following control treatment, 4 h treatment with 50 µg/mL rapamycin (rap) to induce autophagy, and 4 h treatment with 50 µg/mL rapamycin and 10 mM 3-methyladenine (3-MA) to block autophagy. Viability is expressed as % survival relative to the number of viable bacteria in untreated resting control macrophages. Each symbol represents the average of three triplicate infections for each condition using RM sample. The average and standard deviation of all samples are shown. The difference between bacterial survival in control and autophagic macrophages was significant (***, <i>p</i><0.001; *, <i>p</i><0.05; ANOVA).</p

Bactericidal capacity of autophagic macrophages from adult RM.

<p>A. Alveolar macrophages were infected at an MOI of 5∶1 with <i>M. tuberculosis</i> CDC1551. Bacterial colony forming units (cfu) were determined following control treatment, 4 h treatment with 50 µg/mL rapamycin (rap) to induce autophagy, and 4 h treatment with 50 µg/mL rapamycin and 10 mM 3-methyladenine (3-MA) to block autophagy. Viability is expressed as % survival relative to the number of viable bacteria in untreated resting control macrophages. Each symbol represents the average of three triplicate infections for each condition using RM sample. The average and standard deviation of all samples are shown. The difference between bacterial survival in control and autophagic macrophages was significant (**, <i>p</i><0.01; ANOVA). B. Immunofluorescence microscopy was performed on control and autophagic alveolar macrophages infected with fluorescently labeled <i>M. tuberculosis</i> CDC1551 (green). Primary antibodies against either the autophagosomal marker LC3 (top) or the lysosomal marker LAMP (bottom) and DyeLight 594-conjugated secondary antibody were used. Arrows indicate a representative mycobacterium co-localized with LAMP-1. C. The number of <i>M. tuberculosis</i> that co-localize with LAMP-1 were quantified. The difference between control and autophagic cells was not significantly different (p = 0.0586; ANOVA; n = 6 NHP samples).</p

Assessment of autophagy in alveolar macrophages from adult RM.

<p>A. Western analysis was performed on cell lysates from resting control (C) or autophagic (Rap) alveolar macrophages using antibody against LC-3 and actin. The LC-3 antibody reacts with both free cytosolic LC3-I (18 kDa) and membrane associated LC3-II (16 kDa). Levels of LC3-II were normalized to actin by densitometry and the ratio of LC3-II/actin given below the blot. A representative image from a single rhesus macaque (RM) sample is shown. B. Immunofluorescence microscopy was performed on control and rapamycin-treated alveolar macrophages using primary antibody against LC-3. C. The number of cells possessing LC-3+ puncta (top) and the average number of LC3+ vacuoles in LC-3+ cells (bottom) were quantified (n = 6 NHP samples, n>50 macrophages in each condition). The difference between the number of cells possessing LC-3+ puncta was significantly different between control and autophagic cells (***, <i>p</i><0.001; **, <i>p</i><0.01; ANOVA).</p

Bactericidal capacity of resting alveolar macrophages.

<p>Alveolar macrophages were infected at an MOI of 5∶1 with <i>M. tuberculosis</i> CDC1551. Bacterial survival was followed over seven days by harvesting the infected monolayers at the indicated timepoints and plating serial dilutions. The assay was performed in triplicate and the average and standard deviation for each time point are shown for each RM sample. The reference number for each RM is noted in the legend. Bacterial survival in alveolar macrophages from adult (left) and aged (right) RM is shown. There was no significant difference in mycobacterial survival between groups at t = 7d (Student's t-test, <i>p</i> = 0.0693).</p

Impact of menopause and ET on lymphocyte frequencies.

<p>(A) The number of lymphocytes on visit 1 /μl blood based on complete blood cell counts (CBC) values. (B) Frequency of CD4 T cells, CD8 T cells and CD20 B cells was determined in PBMC using flow cytometry (FCM). The percentages were then converted to absolute numbers of cells/μl blood using the lymphocyte counts obtained from the CBCs. (C) Frequency of naïve (Na), central memory (CM) and effector memory (EM) CD4 T cells was determined using FCM and were then converted to number/μl of blood using the CD4 numbers obtained earlier. (D) Numbers of Na, CM and EM CD8 T cells was determined as described for CD4 T cells. (E) Frequencies of naïve (CD27-) and memory (CD27+) B cells were determined by FCM and then converted to absolute numbers of cells/μl blood using the CD20 numbers obtained earlier.</p

Impact of menopause and ET on IL-6 levels and TNFα and IFNγ production.

<p>(A) Frequency of CD4 and CD8 T cells that secrete TNFα, IFNγ or both in response to CD3 stimulation was determined by intracellular cytokine staining and FCM using PBMC collected during visit 1 before the administration of the influenza vaccine. (B) Plasma IL-6 levels using samples collected during visit 1 were determined by ELISA.</p

Optimization of YFV TC-LDA.

<p>In (A), representative flow cytometry dot plots show the results of staining uninfected C6/36 cells or YFV-17D-infected C6/36 cells (MOI = 5, 3 days post-infection) with biotinylated YFV-specific antibodies. Background staining with the secondary reagent, streptavidin-PE (SA-PE) in the absence of a primary YFV-specific antibody was low (<0.004%). Up to 85% of YFV-infected cells could be detected with biotinylated YFV-hyperimmune polyclonal mouse IgG (PAb + SA-PE) but about 98% of YFV-infected C6/36 cells could be detected with biotinylated MAb 3A8.B6 (MAb + SA-PE). In (B), the titer of two strains of YFV (YFV-17D and YFV-Dakar) were determined by TC-LDA (using MAb 3A8.B6) at 1, 3, 7, or 10 days after infection to determine the optimal incubation time required for sensitive quantitation of replicating virus. The data in (B) is based on the average of two independent experiments.</p

Quantitation of yellow fever virus by focus forming assay (FFA).

<p>This representative photograph shows the results of a FFA performed using uninfected Vero cells (top row) or Vero cells infected with YFV-17D (bottom left) or YFV-Dakar (bottom right). At 3 days post-infection, the monolayers were fixed and stained with anti-YFV Mab, 3A8.B6, and infectious foci were identified by addition of detection reagents as described in the methods. No focus forming units (FFU) could be detected in the YFV-Dakar-infected wells at 3, 7, or 10 days after infection with >100 infectious units of virus as determined by TC-LDA. The data is based on 4 experiments, each with similar results.</p

Independent analysis of YFV-infected cells by flow cytometry and RT-PCR.

<p>Dilutions of serum from a YFV-Dakar infected rhesus macaque (day 5 after infection with 1,000 infectious units of YFV-Dakar) were incubated with C6/36 cells (10 wells per dilution) for 10 days prior to removing 10% of the cells from each well for qRT-PCR analysis and then the remaining cells were fixed/stained for YFV antigen as described. The percentage of YFV-positive events shown in each dotplot represents the percentage of cells residing in the gated region that was set based on positive and negative controls included in the assay. Wells were considered positive by flow cytometry if the percentage of positive events were at least 4-fold over the background number of events observed in uninfected wells as indicated by (+) or (−). The titer of YFV-Dakar used in this experiment was calculated to be 4.1×10⁹ infectious units/mL and the sample dilutions performed in these representative dotplots (10⁸, 10⁹, 10¹⁰) is shown on the right-hand side of the figure. Wells that scored positive by YFV RT-PCR (PCR+) or negative by YFV RT-PCR (PCR-) are shown in the figure. There was 100% concordance between these two independent approaches to YFV detection.</p