key: cord-0327326-c1mss66f
authors: Iakobachvili, Nino; Leon Icaza, Stephen Adonai; Knoops, Kèvin; Sachs, Norman; Mazères, Serge; Simeone, Roxane; Peixoto, Antonio; Murris-Espin, Marlène; Mazières, Julien; López-Iglesias, Carmen; Ravelli, Raimond B.G.; Neyrolles, Olivier; Meunier, Etienne; Lugo-Villarino, Geanncarlo; Clevers, Hans; Cougoule, Céline; Peters, Peter J.
title: Mycobacteria-host interactions in human bronchiolar airway organoids
date: 2020-11-12
journal: bioRxiv
DOI: 10.1101/2020.11.12.379586
sha: 4fe176e66be4cac333b3190954f06593d8faa9a5
doc_id: 327326
cord_uid: c1mss66f

Tuberculosis, one of the oldest human pathogens remains a major global health threat. Recent advances in organoid technology offer a unique opportunity to grow different human “organs” in vitro, including the human airway, that faithfully recapitulate tissue architecture and function. We have explored the potential of human airway organoids (AOs) as a novel system in which to model tuberculosis infection. To this end, we adapted biosafety containment level 3–approved procedures to allow successful microinjection of Mycobacterium tuberculosis, the causative agent of tuberculosis, into AOs. We reveal that mycobacteria infected epithelial cells with low efficiency, and that the organoid microenvironment was able to control, but not eliminate the pathogen. We demonstrate that AOs responded to infection by inducing cytokine and antimicrobial peptide production, and inhibiting mucins. Given the importance of myeloid cells in tuberculosis infection, we co-cultured mycobacteria-infected organoids with human monocyte-derived macrophages, and found that these cells were recruited to the organoid epithelium. We conclude that adult stem cell–derived airway organoids can be used to model early events of tuberculosis infection and offer new avenues for fundamental and therapeutic research.

Airborne pathogens are a major cause of death worldwide. Respiratory infectious 54 diseases cause more than 5 million fatalities annually, with tuberculosis (TB) 55 accounting for one-fifth (WHO Global tuberculosis report 2019). In 2018, TB 56 caused an estimated 1.5 million deaths, making TB one of the top 10 killers 57 worldwide, and 25% of the worlds population is thought to be latently infected by 58

Mycobacterium tuberculosis (Mtb) (1). 59

The lung is the entry port for Mtb and the main site of TB disease 60 manifestation. Mtb-containing droplets navigate through the lung anatomy and 61 airway functions in order for mycobacteria to establish its replicative niche in the 62 alveolar space (2, 3). Models of human lung infection are therefore crucial to 63 increase our understanding of host-pathogen interactions-an essential step 64 towards new drug development. Whilst conventional 2D cell culture and animal 65 models have contributed to deciphering key host-pathogen mechanisms at play 66 during Mtb infection (4), they lack relevance with the human lung. 67

One of the major breakthroughs in the stem cell field is the ability to grow 68 human 'organs' in vitro, also known as organoids (5). Human airway organoids 69 (AOs) are derived from adult stem-cells and composed of a polarized, 70 pseudostratified airway epithelium containing basal, secretory and multi-ciliated 71 cells, although they are currently lacking alveolar pneumocytes. They display 72 functional mucus secretion and ciliate beating (6), therefore constituting a suitable 73 human system in which to model early steps of host-pathogen interactions (7-9). 74

We have set out to evaluate the potential of AOs as a model in which to study Mtb 75 infection. Our data demonstrate that mycobacteria can be readily found in the 76 lumen of AOs with some internalization by airway epithelial cells and overall control 77 of mycobacterial growth. In response to Mtb infection, we show AOs inducing the 78 secretion of cytokines and antimicrobial peptides, and the option to model innate 79 cell recruitment by co-culturing human macrophages with injected AOs. 80 81

Due to the innate cystic structure of AOs, where the pathogen-sensing apical side 83 faces the lumen, DsRed-expressing H37Rv Mtb (mean 4271±834 CFU/organoid) 84 was microinjected via a BSL-3-approved custom-made micro-injection system 85 ( Figure 1A ). Bacteria could be found in the lumen of AOs and occasionally making 86 contact with epithelial cells but without causing obvious alterations to organoid 87 architecture and ultrastructure ( Figure 1B -C, Movie S1 and S2). Evident from the 88 movies is the functional mucociliary system where cilia beat secreted mucus and 89 cell debris around the lumen. 90

Mtb is known to infect bronchial epithelial cells in 2D conditions (10), and 91 pneumocytes in vitro (11) and in vivo (12) AOs, which had been infected with Mtb for 7 days, were dissociated into single 99 cells and analyzed by flow cytometry ( Figure 1D ). The functioning mucociliary 100 clearance system within AOs is likely responsible for reducing mycobacterial 101 contact with epithelial cells. 102

Mtb has a functional type VII secretion system (ESX-1) encoded by the RD1 103 locus which is involved in modulating host responses and inducing host cell lysis 104 (13-16). To determine whether the presence of ESX-1 induced increased epithelial 105 cell lysis, we quantified cell death by TOPRO-3 incorporation in AOs after injection 106 of wild-type H37Rv or H37RvESX-1 which lacks ESX-1. Neither strain induced 107 significant epithelial cell death in Mtb-infected AOs compared to uninfected ones 108 (Supp. Figure 1A ), indicating that a functional ESX-1 expression does not trigger 109 increased epithelial cell damage. Organoid culture: AOs were derived from lung biopsies as described (6). was assessed with an ABI 7500 real-time PCR system (Applied Biosystems) and 231 the SYBRTM Select Master Mix (ThermoScientific). Relative quantification was 232 determined using the 2^-Ct method and normalized to GAPDH. Primer 233 sequences are provided in Table 1 . 

The EMBO journal 38(4) . The experiment was performed at least four times independently. ***P < 0.001 by 343 a two-tailed Mann-Whitney test. 344 

The Global Burden of Latent Tuberculosis Infection: A Re-16

4hpi, 77,3% of total cells 24hpi, 25% of total cells

Tables 398 Table 1 . List of primers used for RT-qPCR experiments on airway organoids.