Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis
Thomas R. Lerner, … , Gareth Griffiths, Maximiliano G. Gutierrez
Thomas R. Lerner, … , Gareth Griffiths, Maximiliano G. Gutierrez
Published February 22, 2016
Citation Information: J Clin Invest. 2016;126(3):1093-1108. https://doi.org/10.1172/JCI83379.
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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 6

NO production restricts the growth of cytosolic M. tuberculosis.

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NO production restricts the growth of cytosolic M. tuberculosis. (A) Wes...
(A) Western blot and quantification of the whole-cell protein levels of eNOS and iNOS at 48 hours after infection (normalized to actin) in uninfected and M. tuberculosis WT–infected activated (red bars) or resting (blue bars) hLEC samples. Error bars represent mean ± SEM of 4 biological replicates. (B) Images showing representative areas of M. tuberculosis WT–infected hLECs after 48 hours of infection in activated or resting conditions. Images show bacteria expressing EGFP, endogenous eNOS– or iNOS–Alexa Fluor 546, and DNA labeled with DAPI. Arrows show bacteria and eNOS colocalization. Scale bar: 5 μm. (C) Quantification of the association of eNOS (top panel) and iNOS (bottom panel) with M. tuberculosis WT from images such as those in C at 48 hours after infection. The percentages refers to the proportion of the eNOS+/iNOS+ population (i.e., the population within each dotted box). Error bars represent mean ± SEM from at least 4 biological replicates. **P < 0.01, ***P < 0.001, 2-tailed Student’s t test. (D) The growth (GFP fluorescence per cell) of M. tuberculosis WT, M. tuberculosis ΔRD1, or M. tuberculosis ΔRD1:comp after 48 hours of infection in untreated (control) hLECs or hLECs treated with 250 μM l-NMMA in activated (red symbols) or resting (blue symbols) conditions. Error bars represent mean ± SEM of at least 3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA with Tukey’s post-hoc test. (E) Representative histological section of human lymph nodes stained for PDPN, M. tuberculosis, eNOS, and nuclei (DAPI). The dotted line indicates the granulomatous area. Scale bar: 50 μm. (F) Images of the lymphatic vessel region showing localization of M. tuberculosis in PDPN+ and eNOS+ cells (white arrows). Scale bar: 10 μm.