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Tuberculosis in the age of single cell sequencing

Tuberculosis in the age of single cell sequencing

Tuberculosis is an infectious disease caused by a bacterium Mycobacterium tuberculosis. The bacteria usually attack lungs, but the infection of other parts of the body such as kidney, spine or brain is also possible. Often, an infection shows no symptoms which is also known as latent tuberculosis. In active tuberculosis, the typical symptoms are cough with mucus containing blood, fever, night sweats and weight loss. The spread of bacteria usually happens through the air, when a person with active tuberculosis coughs or speaks. In 2020, an estimated 10 million people developed active tuberculosis, resulting in 1.5 million deaths, making it the second leading cause of death from an infectious disease after COVID-19 (1).

Bacillus Calmette–Guérin (BCG) vaccine is commonly used to vaccinate infants and small children in countries where tuberculosis is common. Among children, it prevents about 20% from getting infected and among those who do get infected, it protects half from developing disease (2). Tuberculosis treatment involves antibiotics, but it is often ineffective due to unusual composition of the cell wall of M. tuberculosis.

We bring you here an overview of recent studies that used single cell sequencing in order to characterize the immune response to M. tuberculosis infection in various research systems.

Characteristics of alveolar macrophages
in tuberculosis patients

Macrophages are specialized immune cells involved in the detection, phagocytosis and destruction of pathogens. In lungs, alveolar macrophages are the first to encounter and help initiate the immune response. Chen and colleagues (3) performed first single cell RNA-seq analysis of alveolar macrophages from bronchioalveolar lavage fluid in active tuberculosis patients and healthy adults. Tuberculosis patients had higher cellular percentage of several macrophage subclusters and also had increased expression of genes involved in inflammatory signaling pathways in the macrophage subclusters. Two novel alveolar macrophage subclusters were identified with distinct functions related to chemokines and adaptive immune response, contradicting the previous report claiming that alveolar macrophages in bronchioalveolar lavage fluid are a homogenous population (4).

Figure 1: Single cell transcriptional profiling of BALF cells. A: UMAP plot of 25 identified clusters. Different colors specify assignment of 25 clusters by nearest neighbor clustering. B: UMAP plot of the 8 cell types annotated by the transcriptional cell markers. (Image from (3),

Single cell analysis of M. tuberculosis phenotype in the infected lung

Pisu and colleagues (5) employed a novel multi-modal approach combining bacterial fitness fluorescent report strains with single cell RNA-seq in a murine model of tuberculosis to associate bacterial and host cell phenotypes at the single cell level. The authors identified macrophage populations with common cell identities across different infection states and characterized those alveolar macrophage subsets that either restricted or promoted bacterial growth. In addition, three distinct populations of interstitial macrophages were identified based on their gene expression. The reported characterization of how host cell populations are regulated before and following infection may facilitate vaccine development and provide new insights in both drug tolerance and acquisition of drug resistance.

Figure 2: Analysis M. tuberculosis phenotype and macrophage populations in the infected lung (Image from (5),

Cellular correlates of tuberculosis control

Lung infection with M. tuberculosis is characterized by granulomas, organized structures derived from host immune and non-imune cells that surround infecting bacteria. Gideon and colleagues (6) applied longitudinal positron emission tomography and computed tomography (PET-CT) imaging, single-cell RNA sequencing, and molecular measures of bacterial killing with immunohistochemistry and flow cytometry to define the cellular ecosystem within tuberculosis lung granulomas. The reported data support a model where high M. tuberculosis burden within granulomas is dictated locally by type 2 immunity and wound-healing responses whereas in granulomas that form later in infection, the balance is tipped toward bacterial control by the emergence of adaptive T1-T17 and cytotoxic responses.

Figure 3: The study was conducted on four cynomolgus macaques infected with a low-dose inoculum of M. tuberculosis. scRNA-seq was performed on 10-week granulomas. (Image from (6),

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1. “Tuberculosis (TB)”. WHO. . Retrieved 21 March2022

2. Roy A, et al.Effect of BCG vaccination against Mycobacterium tuberculosis infection in children: systematic review and meta-analysis. 2014 349: g4643. DOI: 10.1136/bmj.g4643

3. Chen et al. Characteristics of alveolar macrophages in bronchioalveolar lavage fluids from active tuberculosis patients identified by single-cell RNA sequencing. J Biomed Res. 2022 May 28;36(3):167-180. DOI: 10.7555/JBR.36.20220007

4. Mould KJ et al. Airspace macrophages and monocytes exist in transcriptionally distinct subsets in healthy adults. Am J Respir Crit Care Med. 2021;203(8):946–956. doi: 10.1164/rccm.202005-1989OC.

5. Pisu et al. Single cell analysis of M. tuberculosis phenotype and macrophage lineages in the infected lung. J Exp Med. 2021 Sep 6; 218(9): e20210615. doi: 10.1084/jem.20210615

6. Gideon et al. Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control. Immunity 2022 May 10;55(5):827-846.e10. doi: 10.1016/j.immuni.2022.04.004.