TLR7/8/MyD88 trigger inflammatory signaling induced by Mav infection.

<p>Human MDMs were treated with siRNA against target genes or a non-targeted control before infection with Mav-CFP (10 min uptake followed by chase for the indicated times). Cell supernatants were harvested at the indicated time points post infection and secreted cytokines assessed by multiplex ELISA. Cytokine responses from Mav-infected MDMs pretreated with siMyD88 (A), siTLR7, siTLR8, siUNC93B1 or siNOD1 (B). Graphs represent average concentrations +/- SEM of TNF-α, IL-6, IL-10 and IL-8 from at least 6 donors treated with target siRNA (red) or non-targeted siRNA (black). (C) Cells were pre-treated and infected as indicated, fixed and stained for IRF-1 and NF-κB nuclear translocation before analysis using non resolution-limited confocal microscopy. Quantification graphs represent the mean value for each time point (n>700 cells per time point) of one representative donor from three independent experiments. <i>P</i> values between Ctrl and each time point were calculated using Fisher Exact Test (* <i>P</i> <0.05, ** <i>P</i> <0.01 and *** <i>P</i> <0.005.).</p

Mav temporarily resides in the phagolysosomal compartment.

<p>Human MDMs were infected with Mav-CFP (red) for 10 min followed by a chase for 5 minutes to 3 days, stained for EEA1 ((A), early endosomes, green) or LAMP1 ((D, E) late endosomes/lysosomes, green) using antibodies and for nuclei using Hoechst (red), and analyzed by confocal microscopy at the indicated time points. Single labeling (left and middle images) and merged images (right images) are shown. Bottom-to-top projections of 3D-stacks from boxed areas are shown in lower panels (left to right) and represent Mav, EEA1/LAMP1⁺ membranes and merged images. Quantification of Mav localization in EEA1⁺ or LAMP1⁺ compartments was performed on 3D stacks using fluorescence intensity profiles along the indicated line (<i>FAL</i>, (B) and (F), Mav: red line; EEA1/LAMP1: green line). For each time point, at least 70 cells were recorded per donor. Quantification graphs represent mean value +/- SEM of Mav localization in EEA1⁺ (C) or LAMP1⁺ (G) compartments for 3 donors. <i>P</i> values were calculated using Fisher Exact Test (* <i>P</i> <0.05, ** <i>P</i> <0.01 and *** <i>P</i> <0.005). a.u.: arbitrary unit. Scale bar represents 10 μm.</p

TLR7/8/MyD88 regulate intracellular growth of Mav in human primary macrophages.

<p>Human MDMs were treated with siRNA against MyD88, TLR7, TLR8 or a non-targeting control before infection with Mav-CFP (10 min uptake followed by 4h to 3d chase). (A) Representative images where infected cells treated with siNTC (left) or siTLR8 (right) are outlined for analysis (Mav-CFP: red, nuclei: blue (Hoechst)). (B, C) <i>In situ</i> quantification of Mav-CFP 4 hours (B) and 3 days (C) post infection using the Corrected Total Cell Fluorescence method on infected cells. Quantification scatter plots show individual cell measures with mean +/- 95% confident intervals from 3 donors ((B, C) left graphs, n>250 cells per time point and per donor). The bar chart in (B) shows the percentage of infected cells 4 hours post infection to compare uptake. (D) Working model. Mav (dashed line, blue) is processed in LAMP1⁺ phagolysosomes. Release of mycobacterial nucleic acids engages TLR7/8, recruitment of MyD88 to the compartment, and inflammatory signaling culminating in cytokine release and growth restriction. A fraction of live Mav actively remodels the phagolysosome and/or is sorted into a new compartment (MavC) where late endosomal/lysosomal markers are excluded, TLR7/8 are not engaged and MyD88 is not recruited, thus avoiding inflammatory signaling and destruction. The program leading to Mav destruction (red line) remains to be elucidated.</p

Activation of IRF-1 coincides with phagolysosomal localization of Mav.

<p>Human MDMs were infected with live or PFA-killed Mav-CFP (red) for 10 min, chased for 4h to 3d and stained with antibodies to IRF-1 (blue) and LAMP1 (green) before analysis of MDMs containing only single Mav using confocal microscopy. Nuclei were revealed by staining with Hoechst (red). Single and merged images are shown; bottom-to-top projections of 3D-stacks from boxed areas in lower panels represent Mav, LAMP1 and merged images (A, C). (A) Cell with live Mav in LAMP1⁺ compartment inducing nuclear translocation of IRF-1. (C) Cell with live Mav in LAMP1^- compartment and no nuclear translocation of IRF-1. (B, D) Quantification of live Mav localization in LAMP1⁺ compartments and nuclear localization of IRF-1 was performed on 3D stacks using fluorescence intensity profiles along the indicated lines of Mav phagosomes ((B) and (D), left (Mav: red trace; LAMP1: green trace) and right (Hoechst: red trace; IRF-1: green trace) graphs respectively). (E) The fraction of live Mav localized to LAMP1⁺ phagolysosomes in IRF-1-activated cells (mean percentage +/- SEM from 3 donors). For each time point, at least 70 cells were recorded for each donor. (F) IRF-1 nuclear translocation at indicated time points after challenge with live or PFA-killed Mav. Quantification graphs represent the mean value +/- SEM for each time point for live Mav (black bars) and PFA-killed Mav (red bars) (n>600 cells per time point and per donor, 3 donors). <i>P</i> values between live Mav and PFA-killed Mav were calculated using two-tailed t-test (* <i>P</i> <0.05, ** <i>P</i> <0.01 and *** <i>P</i> <0.005.). a.u.: arbitrary unit. Scale bar represents 10 μm.</p

Phagolysosomes retain PFA-killed Mav.

<p>Human MDMs were exposed to PFA-killed Mav-CFP (10 min uptake, chased for 4h to 3d), stained with anti-LAMP1 antibody and Hoechst (nucleus) and analyzed using resolution-limited confocal microscopy. (A) LAMP1⁺ late endosomes/lysosomes (green), Mav and nuclei (red). Single labeling (left and middle images) and merged images (right images) are shown. Bottom-to-top projections of 3D-stack from boxed area are shown in lower panels and represent Mav, LAMP1⁺ membranes and merged images. Quantification of Mav localization in LAMP1⁺ compartments was performed on 3D stacks using fluorescence intensity profiling of Mav phagosomes along the indicated line ((B), Mav: red line; LAMP1: green line). For each time point, at least 70 cells were recorded for each donor. (<b>c</b>) The percentage of Mav localized to LAMP1⁺ compartments (mean value +/- SEM for 3 donors). <i>P</i> values were calculated using two-tailed t-test (* <0.05, ** <0.01 and *** <0.005.). a.u.: arbitrary unit. Scale bar represents 10 μm.</p

TNF production coincides with phagolysosomal localization of Mav.

<p>Human MDMs were infected with live Mav-CFP (blue) for 10 min, chased for 4h in the presence of a protein secretion inhibitor to accumulate cytokines, fixed and stained with antibodies to LAMP1 (red) and TNF (green). (A) Single and merged images of one cell; bottom-to-top projections of 3D-stack from boxed area in lower panels show that Mav is in a LAMP1⁺ compartment. (B) Fluorescence intensity profiles along the indicated lines of the Mav phagosome (Mav: blue trace; LAMP1: red trace). (C) Quantification of the percentage of infected cells secreting TNF (black bar), and of these, the fraction with Mav in LAMP1⁺ compartments (red bar) or LAMP1^- compartments (blue bar). Quantification graphs represent the mean value +/- SEM from two different experiments. (D) Human MDMs were treated with siMyD88, siTLR8 or non-targeted control before infection with Mav for 10 min and chase for 4h. Cells were then stained with antibody to MyD88 and analyzed using confocal microscopy for recruitment of MyD88 to Mav phagosomes. Quantification graph represents MyD88-Mav association from two independent experiments. Scale bar represents 10 μm.</p

MavCs, but not MavPLs, support Mav replication.

<p>Human MDMs were infected with Mav-CFP for 10 min, chased for 5 min to 3d, fixed with 4% PFA and analyzed by confocal microscopy. <i>In situ</i> quantification of the actual number of Mav inside (left panel) or outside (right panel) LAMP1⁺ compartments at different time points, including the mean value +/- 95% confidence intervals.</p