Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 May;12(3):795-804.
doi: 10.1038/s41385-019-0156-2. Epub 2019 Mar 7.

The human lung mucosa drives differential Mycobacterium tuberculosis infection outcome in the alveolar epithelium

J M Scordo  1   2 , A M Olmo-Fontánez  2 , H V Kelley  1   2 , S Sidiki  1 , J Arcos  1 , A Akhter  2 , M D Wewers  3 , J B Torrelles  4   5
Affiliations

The human lung mucosa drives differential Mycobacterium tuberculosis infection outcome in the alveolar epithelium

J M Scordo et al. Mucosal Immunol. 2019 May.

Abstract

Mycobacterium tuberculosis (M.tb) is deposited into the alveolus where it first encounters the alveolar lining fluid (ALF) prior contacts host cells. We demonstrated that M.tb-exposure to human ALF alters its cell surface, driving better M.tb infection control by professional phagocytes. Contrary to these findings, our results with non-professional phagocytes alveolar epithelial cells (ATs) define two distinct subsets of human ALFs; where M.tb exposure to Low (L)-ALF or High(H)-ALF results in low or high intracellular bacterial growth rates in ATs, respectively. H-ALF exposed-M.tb growth within ATs was independent of M.tb-uptake, M.tb-trafficking, and M.tb-infection induced cytotoxicity; however, it was associated with enhanced bacterial replication within LAMP-1+/ABCA1+ compartments. H-ALF exposed-M.tb infection of ATs decreased AT immune mediator production, decreased AT surface adhesion expression, and downregulated macrophage inflammatory responses. Composition analysis of H-ALF vs. L-ALF showed H-ALF with higher protein tyrosine nitration and less functional ALF-innate proteins important in M.tb pathogenesis. Replenishment of H-ALF with functional ALF-innate proteins reversed the H-ALF-M.tb growth rate to the levels observed for L-ALF-M.tb. These results indicate that dysfunctionality of innate proteins in the H-ALF phenotype promotes M.tb replication within ATs, while limiting inflammation and phagocyte activation, thus potentiating ATs as a reservoir for M.tb replication and survival.

PubMed Disclaimer

Conflict of interest statement

DISCLOSURE

The authors declared no conflict of interest.

Figures

Figure 1.
Figure 1.. Human alveolar lining fluid drives M.tb growth in ATs.
(A) ALF-exposed M.tb intracellular growth in ATs 5 days post-infection (DPI) was assessed by colony forming units (CFUs) for determination of intracellular M.tb. Data were normalized to AT cell count. Numbers 1–14 represent ALFs, each from a different healthy human donor. Numbers between graphs correspond to the same ALF. Errors bars represent technical replicates (all ALFs were performed in replicate within the same experiment) and/or multiple experiments (for ALFs 5, 6, 9, 11, and 13). (B) Exposed-M.tb growth rate calculated as slope of intracellular M.tb growth throughout 5 day AT infection. Growth rate is an average value obtained from replicate experiments of n=3 for ALFs 11 & 13, n=2 for ALFs 2, 4, 5, 6, 9, 12 & 14 and for ALFs 1, 3, 7, 8, & 10 n=1. L defined as Low Growth (L)-ALF-M.tb (white bars) and H defined as High Growth (H)-ALF-M.tb (black bars) with the difference in growth rate >0.065; Student’s t test L-ALF vs. High-ALF ****p<0.0001. (C) Linear regression of ALF-M.tb uptake (2 h post-infection, x-axis) vs ALF-M.tb intracellular growth at 5 DPI (y-axis). R2 = 0.002. Low-ALF (white-circles), High-ALF (black circles), and mid-ALFs (grey circles). (D) M.tb exposed to L-ALF, H-ALF, or left unexposed was serially diluted in 7H9 broth and plated on 7H11 agar plates for determination of M.tb viability (extracellular growth) by CFUs. Shown are individual exposed-M.tb with n=4 L-ALF (white bars), n=5 H-ALF (black bars), and n=4 unexposed-M.tb (U, grey bars), with combined values shown in the graph on right. Freshly ALF exposed-M.tb were always used immediately for AT infections for determination of ALF exposed-M.tb growth rates in ATs.
Figure 2.
Figure 2.. L-ALF and H-ALF does not alter M.tb intracellular trafficking within ATs.
GFP-M.tb co-localization with intracellular markers by confocal microscopy at 3 DPI. (A) Representative confocal images showing M.tb exposed to Low ALF (top panels) or High ALF (bottom panels) co-localization with LAMP-1 (red) and LC3 (light blue). Region in white dashed-line box is shown on the right with merged events indicated by white arrowheads. (B) Quantification of LAMP-1+M.tb co-localization events for n=3 Low ALFs and n=4 High ALFs. (C) Quantification of LC3+M.tb co-localization events for n=2 Low and High ALFs. (D) Representative confocal images of Low- (top panels) and High-ALF-M.tb (bottom panels) co-localization with ABCA1 (purple) and ABCA3 (red) at 3 DPI. Region in white dashed-line box is shown on the right with merged events indicated by white arrowheads. (E) Quantification of ABCA1+M.tb co-localization events for n=3 Low ALFs and n=4 High ALFs. (F) Quantification of ABCA3+M.tb co-localization events for n=3 Low and High ALFs. (G) Representative confocal images showing ALF-exposed M.tb (top panels, L-ALF; bottom panels, H-ALF) co-localization with LysoTracker in infected ATs at 3 DPI. White dashed-line box region is shown on the right, with merged events indicated by white arrowheads. (H) Quantification of LysoTracker co-localization with n=2 Low ALF-M.tb and n=3 High ALF-M.tb. All co-localization experiments were quantified by counting >100 events per coverslip, in replicate.
Figure 3.
Figure 3.. High-ALF-M.tb has increased rate of replication during AT infection and favors AT MVBs and late endosomes.
ATs were infected with SSB-GFP, smyc’∷mCherry M.tb for 3 days and M.tb replication rate was determined by confocal microscopy. (A) Representative confocal images of ATs infected with Low- and High-ALF-M.tb. Region indicated by white dashed-line is shown on right (L-ALF, top panels and H-ALF, bottom panels). SSB+ M.tb are indicated by white arrowheads, showing merged (yellow) foci. (B) Percentage of SSB+ M.tb exposed to low and high ALFs at 3 DPI was quantified by counting >100 events per coverslip, in replicate (M±SD). N=2 L and 2 H ALFs; Student’s t test L-ALF vs. High-ALF *p<0.05. (C) Representative confocal images of ATs infected with High-ALF-M.tb. Region indicated by white dashed-line is shown on the right. White arrows indicate SSB+ M.tb co-localization with respective intracellular AT marker. (D) Percentage of SSB+ H-ALF exposed M.tb co-localized with the following: ABCA1, ABCA3, LC3 and LAMP-1. Each dot represents M.tb exposed to a H-ALF in replicate, quantified by counting >25 events per coverslip (M±SEM). One-way ANOVA post-Tukey analysis; for LAMP-1 vs ABCA3 & LAMP-1 vs LC3 *p<0.05.
Figure 4.
Figure 4.. H-ALF-M.tb induces less cell death and decreases AT immune mediators.
ATs were infected with L-ALF and H-ALF-M.tb for 5 days for determination of AT cell death (A&B) or AT immune mediator production (C&D) and expression (E&F). (A) ATs were assessed for early and late apoptosis/necrosis by flow cytometry using Annexin V & 7-AAD live/dead staining per kit instructions. Representative flow cytometry plots shown on left and quantification on right. Experiment performed in duplicate (mean ± SEM); n=4 Low ALFs & 3 High ALFs. (B) AT supernatants were tested for % cytotoxicity by release of LDH (measure of necrosis), per kit instructions; n=7 L-ALFs & 5 H-ALFs in duplicate (mean ± SEM). (C) AT chemokine production in cell supernatants measured by ELISA per kit instructions. N=4 L-ALFs and 4 H-ALFs in triplicate; mean ± SEM. (D) AT cytokine production in cell supernatants tested by Multiplex Assay per kit instructions. N= 4 L-ALFs and 4 H-ALFs in duplicate; mean ± SD. (E) AT mRNA expression measured by qRT-PCR and shown as relative fold change versus uninfected ATs. N=3 Low and 3 High ALFs, in duplicate; mean ± SEM. (F) Mean Fluorescence Intensity (MFI) of AT cell surface expression of ICAM-1 assessed by flow cytometry. N=2 Low and 2 High ALFs, in replicate; mean ± SEM. L=L-ALF-M.tb, H=H-ALF-M.tb, U = uninfected ATs & PC = ethanol-treated ATs. Student’s t test, L-ALF- vs. H-ALF-M.tb; *p<0.05, **p<0.01, ***p<0.001.
Figure 5.
Figure 5.. H-ALF-M.tb infected ATs modulate human macrophage immune response.
ATs were infected with L- or H-ALF-M.tb for 5 days and (A) exposed-M.tb infected ATs were co-cultured with primary human monocyte-derived macrophages (MDMs) or (B) exposed-M.tb infected AT supernatant was 0.2 μm-sterile filtered and incubated with MDMs. After 24 hours, macrophage protein production was assessed by ELISA per kit instructions to determine (A) direct and (B) indirect modulation of MDM cytokine release by exposed-M.tb ATs. N=3 Low and 3 High ALFs, performed in duplicate (mean ± SEM) with 2 different MDM donors. L= resting MDMs incubated with L-ALF-M.tb infected ATs (or supernatant), H= resting MDMs incubated with H-ALF-M.tb infected ATs (or supernatant), U= resting MDMs incubated with uninfected ATs or (supernatant). Student’s t test, L-ALF- vs. H-ALF-M.tb; **p<0.01. ND = not determined.
Figure 6.
Figure 6.. H-ALF- proteins have increased tyrosine nitration, are less functional, and drive enhanced H-ALF exposed-M.tb growth rate in ATs.
Levels of oxidative stress in Low- (L) and High- (H) ALF samples were detected by ELISA for presence of (A) 3-nitrotyrosine residues, (B) carbonyls or (C) myeloperoxidase (MPO). All ALFs were run in duplicate for n=4, mean ± SD. Student’s t test, L-ALF vs. H-ALF; **p<0.01. Innate protein function, measured as the ability of innate proteins to bind to the M.tb surface, was determined using indirect ELISA after M.tb exposure to L- or H-ALF for 12 hours (D-F). Binding assays were performed in triplicate and calculated as the absorbance (OD 450nm) vs. positive control (human SP-A protein (3 μg), human SP-D protein (1 μg) & human C3 protein (10 μg). Innate protein function (y-axis) is plotted as a function of ALF subset (Low ALF, shown as white circles and High ALF, as black circles) and 3-nitrotyrosine content (x-axis) for (D) C3, (E) SP-A and (F) SP-D. Shown is n=4, mean ± SD. All data shown has been normalized for 1 mg/mL ALF phospholipid content. (G and H) M.tb was exposed to Low ALF, High ALF, or L-ALF and H-ALF replenished with the following recombinant human proteins for 12 hours: C3 (+C3, 20 μg/mL), SP-A (+SP-A, 10 μg/mL), SP-D (+SP-D, 5 μg/mL), all 3 combined (+ALL), or human serum albumin (40 μg/mL) as an irrelevant protein control (+ALB). Following the 12-hour incubation, exposed-M.tb were pelleted, washed to remove traces of ALF and unbound proteins, and used to infect ATs at MOI 10:1 for 2 hours. Low ALFs are shown in white bars and High ALFs shown in black bars. (G) AT-exposed-M.tb uptake was assessed after 2-hour infection and 1-hour gentamicin treatment for determination of intracellular CFUs. (H) Exposed-M.tb growth rate (slope of M.tb intracellular growth in ATs) was determined throughout 5 days of intracellular AT infection. Shown is n=4 Low and 4 High ALFs (ALFs without replenishment) and n=2 Low and 2 High ALFs for respective ALF-replenishment studies, mean ± SEM. One-way ANOVA post-Dunnett’s analysis L-ALF vs. H-ALF **p<0.01 and for replenished H-ALF groups vs. H-ALF ##p<0.01, ###p<0.001, ####p<0.0001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Erck D Take 1.5 minutes for 1.5 million lives. (Aeras, 2016). www.aeras.org
    1. Torrelles JB & Schlesinger LS Integrating lung physiology, immunology, and tuberculosis. Trends Microbiol 25, 688–697 (2017). - PMC - PubMed
    1. Sia JK, Georgieva M, Rengarajan J Innate immune defenses in human tuberculosis: An overview of the interactions between Mycobacterium tuberculosis and innate immune cells. Journal of immunology research 2015, 12 (2015). - PMC - PubMed
    1. Arcos J, et al. Mycobacterium tuberculosis cell wall released fragments by the action of the human lung mucosa modulate macrophages to control infection in an IL-10-dependent manner. Mucosal Immunol 10, 1248–1258 (2017). - PMC - PubMed
    1. Scordo JM, et al. Mycobacterium tuberculosis cell wall fragments released upon bacterial contact with the human lung mucosa alter the neutrophil response to infection. Front Immunol 8, 307 (2017). - PMC - PubMed

Publication types

-