Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis
Thomas R. Lerner, Cristiane de Souza Carvalho-Wodarz, Urska Repnik, Matthew R.G. Russell, Sophie Borel, Collin R. Diedrich, Manfred Rohde, Helen Wainwright, Lucy M. Collinson, Robert J. Wilkinson, Gareth Griffiths, Maximiliano G. Gutierrez
Thomas R. Lerner, Cristiane de Souza Carvalho-Wodarz, Urska Repnik, Matthew R.G. Russell, Sophie Borel, Collin R. Diedrich, Manfred Rohde, Helen Wainwright, Lucy M. Collinson, Robert J. Wilkinson, Gareth Griffiths, Maximiliano G. Gutierrez
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Research Article Cell biology Infectious disease

Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis

Abstract

In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes.

Authors

Thomas R. Lerner, Cristiane de Souza Carvalho-Wodarz, Urska Repnik, Matthew R.G. Russell, Sophie Borel, Collin R. Diedrich, Manfred Rohde, Helen Wainwright, Lucy M. Collinson, Robert J. Wilkinson, Gareth Griffiths, Maximiliano G. Gutierrez

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Figure 3

Subcellular localization of M. tuberculosis reveals a heterogeneous localization in hLECs.

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Subcellular localization of M. tuberculosis reveals a heterogeneous loca...
(A) Representative images of hLECs infected with M. tuberculosis WT, M. tuberculosis ΔRD1, or M. tuberculosis ΔRD1:comp at 48 hours after infection in the presence or absence of IFN-γ. Images show bacteria expressing EGFP, endogenous cathepsin D–Alexa Fluor 546, F-actin labeled with Alexa Fluor 633–phalloidin, and DNA labeled with DAPI. Scale bar: 10 μm. Original magnification, ×5 (inset). (B) Quantification of the association of cathepsin D with M. tuberculosis WT, M. tuberculosis ΔRD1, or M. tuberculosis ΔRD1:comp from images such as those in A at 48 hours after infection. N is the total number of individual bacterial entities measured in each condition, and the percentages refer to the proportion of the cathepsin D+ population (i.e., the population within each dotted box). Error bars represent mean ± SEM from 3 biological replicates. ***P < 0.001, 1-way ANOVA with Tukey’s post-hoc test. (C) Example TEM images representing the observed localization of M. tuberculosis WT, M. tuberculosis ΔRD1, or M. tuberculosis ΔRD1:comp in hLECs at 48 hours after infection. “Phagosome” represents bacteria with a single surrounding host membrane; “Cytosol” represents bacteria with no surrounding host membranes; “Autophagosome” represents bacteria found with 2 or more surrounding host membranes; and “Lysosome” represents surrounded by a host membrane filled with vesicles. Scale bar: 500 nm. (D) Quantification of M. tuberculosis WT, M. tuberculosis ΔRD1, or M. tuberculosis ΔRD1:comp subcellular localizations within hLECs at 48 hours after infection with in activated (red bars) or resting (blue bars) cells. Quantification was performed by stereological analysis of TEM images of resin sections of hLECs. Error bars represent mean ± SEM from at least 2 biological replicates.