Characterization of host and microbial determinants in individuals with latent tuberculosis infection using a human granuloma model

Abstract

Granulomas sit at the center of tuberculosis (TB) immunopathogenesis. Progress in biomarkers and treatment specific to the human granuloma environment is hindered by the lack of a relevant and tractable infection model that better accounts for the complexity of the host immune response as well as pathogen counterresponses that subvert host immunity in granulomas. Here we developed and characterized an in vitro granuloma model derived from human peripheral blood mononuclear cells (PBMCs) and autologous serum. Importantly, we interrogated this model for its ability to discriminate between host and bacterial determinants in individuals with and without latent TB infection (LTBI). By the use of this model, we provide the first evidence that granuloma formation, bacterial survival, lymphocyte proliferation, pro- and anti-inflammatory cytokines, and lipid body accumulation are significantly altered in LTBI individuals. Moreover, we show a specific transcriptional signature of Mycobacterium tuberculosis associated with survival within human granuloma structures depending on the host immune status. Our report provides fundamentally new information on how the human host immune status and bacterial transcriptional signature may dictate early granuloma formation and outcome and provides evidence for the validity of the granuloma model and its potential applications.

Importance: In 2012, approximately 1.3 million people died from tuberculosis (TB), the highest rate for any single bacterial pathogen. The long-term control of TB requires a better understanding of Mycobacterium tuberculosis pathogenesis in appropriate research models. Granulomas represent the characteristic host tissue response to TB, controlling the bacilli while concentrating the immune response to a limited area. However, complete eradication of bacteria does not occur, since M. tuberculosis has its own strategies to adapt and persist. Thus, the M. tuberculosis-containing granuloma represents a unique environment for dictating both the host immune response and the bacterial response. Here we developed and characterized an in vitro granuloma model derived from blood cells of individuals with latent TB infection that more accurately defines the human immune response and metabolic profiles of M. tuberculosis within this uniquely regulated immune environment. This model may also prove beneficial for understanding other granulomatous diseases.

FIG 1

In vitro granuloma-like structures are formed by M. tuberculosis infection of PBMCs from LTBI individuals. (a) PBMCs obtained from LTBI individuals were infected with M. tuberculosis H₃₇R_v (MOI 1:1), resulting in the formation of granulomas by day 7 postinfection. (b) Higher magnification of the granulomas by confocal microscopy. (c) No formation of granulomas was observed in uninfected PBMCs obtained from LTBI individuals for up to 12 days postinfection. (d to f) Confocal microscopy images of the granulomas at day 7 postinfection revealed multicellular, multilayered structures containing approximately 4 to 8 cell layers. (d) Differential inference contrast (DIC) image. (e) Image of transverse and straight sections by orthogonal view. (f) Three-dimensional (3D) view image. Nuclei were stained with DAPI (cyan). (g) Granuloma-like structures include macrophages (CD11b⁺, red) and T cells (CD3⁺, green). (h) Confocal microscopy images of the granulomas at day 7 postinfection revealed the presence of multinucleated giant cells (CD11b⁺⁺, yellow; white arrow). Nuclei were stained with DAPI (dark blue). Representative images from n = 12 experiments are shown. The images in panels a and c are shown with ×40 magnification; the remaining images are ×60.

FIG 2

Granuloma-like structure formation is accelerated in cells from individuals with LTBI. (a) PBMCs obtained from LTBI and naive individuals were infected with M. tuberculosis H₃₇R_v (MOI 1:1) for up to 12 days. While naive individuals typically demonstrated no evidence of cell aggregation until day 6 postinfection, on average, the LTBI cell cultures exhibited phase 3 to 4. (b) The scoring system was applied on day 7 postinfection, and 15 high-power fields/sample were evaluated. Naive individuals, light grey bars; LTBI individuals, dark grey bars. All images are ×40. Uninfected cells were used as controls. n = 12 in triplicate. ***, P < 0.001 (by t test). M.tb, M. tuberculosis.

FIG 3

Granuloma-like structures from LTBI individuals have better control of M. tuberculosis growth over time. PBMCs obtained from LTBI and naive individuals were infected with M. tuberculosis R_v-lux (MOI 1:1) for up to 9 days. (a and b) RLUs (a) and CFU (b) were determined at several time points after infection. (c) Confocal microscopy images of the granulomas at day 7 postinfection revealed more bacilli (green) in granuloma-like structures from naive individuals than in those from LTBI individuals. Naive individuals, light grey bars; LTBI individuals, dark grey bars. Uninfected cells were used as controls. All images are ×60. n = 3 in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by two-way ANOVA with Bonferroni posttest).

FIG 4

The proliferative activity of granuloma-like structures from LTBI individuals is significantly greater than that seen with naive individuals. PBMCs obtained from LTBI and naive individuals were infected with M. tuberculosis H₃₇R_v (MOI 1:1) for up to 7 days, and cell proliferation was determined by EdU assay. (a) Confocal microscopy images of the granulomas at day 7 postinfection revealed greater proliferation (green) in granulomas from LTBI individuals than in those from naive individuals. (b) Quantification of the cellular fluorescence shown by calculation of MFI values. Naive individuals, light grey bars; LTBI individuals, dark grey bars. Uninfected cells were used as controls. All images are ×40. n = 3. **, P < 0.01 (by t test).

FIG 5

Granuloma-like structures from LTBI individuals produce more robust inflammatory cytokines. PBMCs obtained from LTBI and naive individuals were infected with M. tuberculosis H₃₇R_v (MOI 1:1) for up to 12 days, and cytokine levels in cell culture supernatants collected at several time points after infection were determined by ELISA. Naive individuals, light grey bars; LTBI individuals, dark grey bars. Uninfected cells were used as controls. ND, not detected. n = 3 (in triplicate). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by t test).