ESX1-dependent fractalkine mediates chemotaxis and Mycobacterium tuberculosis infection in humans

SummaryMycobacterium tuberculosis-induced cellular aggregation is essential for granuloma formation and may assist establishment and early spread of M. tuberculosis infection. The M. tuberculosis ESX1 mutant, which has a non-functional type VII secretion system, induced significantly less production of the host macrophage-derived chemokine fractalkine (CX3CL1). Upon infection of human macrophages ESX1-dependent fractalkine production mediated selective recruitment of CD11b+ monocytic cells and increased infection of neighbouring cells consistent with early local spread of infection. Fractalkine levels were raised in vivo at tuberculous disease sites in humans and were significantly associated with increased CD11b+ monocytic cellular recruitment and extent of granulomatous disease. These findings suggest a novel fractalkine-dependent ESX1-mediated mechanism in early tuberculous disease pathogenesis in humans. Modulation of M. tuberculosis-mediated fractalkine induction may represent a potential treatment option in the future, perhaps allowing us to switch off a key mechanism required by the pathogen to spread between cells

Enumeration of Functional T-Cell Subsets by Fluorescence-Immunospot Defines Signatures of Pathogen Burden in Tuberculosis

IFN-γ and IL-2 cytokine-profiles define three functional T-cell subsets which may correlate with pathogen load in chronic intracellular infections. We therefore investigated the feasibility of the immunospot platform to rapidly enumerate T-cell subsets by single-cell IFN-γ/IL-2 cytokine-profiling and establish whether immunospot-based T-cell signatures distinguish different clinical stages of human tuberculosis infection.We used fluorophore-labelled anti-IFN-γ and anti-IL-2 antibodies with digital overlay of spatially-mapped colour-filtered images to enumerate dual and single cytokine-secreting M. tuberculosis antigen-specific T-cells in tuberculosis patients and in latent tuberculosis infection (LTBI). We validated results against established measures of cytokine-secreting T-cells.Fluorescence-immunospot correlated closely with single-cytokine enzyme-linked-immunospot for IFN-γ-secreting T-cells and IL-2-secreting T-cells and flow-cytometry-based detection of dual IFN-γ/IL-2-secreting T-cells. The untreated tuberculosis signature was dominated by IFN-γ-only-secreting T-cells which shifted consistently in longitudinally-followed patients during treatment to a signature dominated by dual IFN-γ/IL-2-secreting T-cells in treated patients. The LTBI signature differed from active tuberculosis, with higher proportions of IL-2-only and IFN-γ/IL-2-secreting T-cells and lower proportions of IFN-γ-only-secreting T-cells.Fluorescence-immunospot is a quantitative, accurate measure of functional T-cell subsets; identification of cytokine-signatures of pathogen burden, distinct clinical stages of M. tuberculosis infection and long-term immune containment suggests application for treatment monitoring and vaccine evaluation

Innate activation of human primary epithelial cells broadens the host response to Mycobacterium tuberculosis in the airways

Early events in the human airways determining whether exposure to Mycobacterium tuberculosis (Mtb) results in acquisition of infection are poorly understood. Epithelial cells are the dominant cell type in the lungs, but little is known about their role in tuberculosis. We hypothesised that human primary airway epithelial cells are part of the first line of defense against Mtb-infection and contribute to the protective host response in the human respiratory tract. We modelled these early airway-interactions with human primary bronchial epithelial cells (PBECs) and alveolar macrophages. By combining in vitro infection and transwell co-culture models with a global transcriptomic approach, we identified PBECs to be inert to direct Mtb-infection, yet to be potent responders within an Mtb-activated immune network, mediated by IL1β and type I interferon (IFN). Activation of PBECs by Mtb-infected alveolar macrophages and monocytes increased expression of known and novel antimycobacterial peptides, defensins and S100-family members and epithelial-myeloid interactions further shaped the immunological environment during Mtb-infection by promoting neutrophil influx. This is the first in depth analysis of the primary epithelial response to infection and offers new insights into their emerging role in tuberculosis through complementing and amplifying responses to Mtb

Stratification of Latent Mycobacterium tuberculosis Infection by Cellular Immune Profiling

This work was supported by the United Kingdom National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Respiratory Infections, as well as a Wellcome Trust Senior Clinical Research Fellowship and a NIHR Senior Investigator Award (both awarded to A. L.)

Undetected multidrug-resistant tuberculosis amplified by first-line therapy in mixed infection

Infections with >1 Mycobacterium tuberculosis strain(s) are underrecognized. We show, in vitro and in vivo, how first-line treatment conferred a competitive growth advantage to amplify a multidrug-resistant M. tuberculosis strain in a patient with mixed infection. Diagnostic techniques that identify mixed tubercle bacilli populations are needed to curb the spread of multidrug resistance

Epithelial cells are the major subset in the airway lining and are not permissive to Mtb infection.

<p>(A) Presence of total bronchial epithelial cells (BECs) and leukocytes (LEU) was measured <i>ex vivo</i> by differential cell counts of bronchial brushings derived from the airway lining. Subsets are shown as % of total cells recovered from healthy volunteers (n = 17). Wilcoxon signed rank test was used to compare groups. Boxplots show median and range. **** p<0.0001. (B) PBECs (n = 4) and THP-1 MΦ (mean ± SD of 2 independent experiments) were infected with Mtb H37Rv for 24h. To remove extracellular bacteria, 200 μg/ml amikacin was added for an additional 2h where indicated. Mtb bacilli were enumerated by colony forming units (CFU). (C) Affymetrix HTA2.0 arrays were performed on PBECs (uninfected, MOI10 or MOI50, 24h) from 4 donors. The log₂ fold change compared to unstimulated PBECs and the associated FDR-adjusted q-value are shown in volcano plots for MOI10 (left) and MOI50 (right). The solid black lines intersect at q-value of 0.05.</p

PBECs express antimycobacterial effectors <i>DEFB4</i> and <i>S100A7</i>. Mtb H37Rv was grown in the presence of the indicated concentrations of recombinant (A) hBD2, (B) psoriasin or vehicle control (veh).

<p>Amikacin and gentamycin were used as positive controls at 200 μg/ml and 100 μg/ml respectively. Mtb growth was measured by optical density at 595nm (OD_595nm) over time. Mean readings of one representative experiment are shown. At day 7, cultures were plated to measure the bacterial burden by CFU (n = 3 representative for two independent H37Rv cultures). Groups were compared against vehicle control at day 7 by one-way ANOVA or Mann-Whitney test (pooled normalised CFU data from three (psoriasin) or two (hBD2) independent experiments are shown). Median is shown. ϕ, p<0.001;**, p<0.01; ***, p<0.001.</p