Serine protease activity contributes to control of Mycobacterium tuberculosis in hypoxic lung granulomas in mice
Stephen T. Reece, Christoph Loddenkemper, David J. Askew, Ulrike Zedler, Sandra Schommer-Leitner, Maik Stein, Fayaz Ahmad Mir, Anca Dorhoi, Hans-Joachim Mollenkopf, Gary A. Silverman, Stefan H.E. Kaufmann
Stephen T. Reece, Christoph Loddenkemper, David J. Askew, Ulrike Zedler, Sandra Schommer-Leitner, Maik Stein, Fayaz Ahmad Mir, Anca Dorhoi, Hans-Joachim Mollenkopf, Gary A. Silverman, Stefan H.E. Kaufmann
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Research Article Infectious disease

Serine protease activity contributes to control of Mycobacterium tuberculosis in hypoxic lung granulomas in mice

Abstract

The hallmark of human Mycobacterium tuberculosis infection is the presence of lung granulomas. Lung granulomas can have different phenotypes, with caseous necrosis and hypoxia present within these structures during active tuberculosis. Production of NO by the inducible host enzyme NOS2 is a key antimycobacterial defense mechanism that requires oxygen as a substrate; it is therefore likely to perform inefficiently in hypoxic regions of granulomas in which M. tuberculosis persists. Here we have used Nos2–/– mice to investigate host-protective mechanisms within hypoxic granulomas and identified a role for host serine proteases in hypoxic granulomas in determining outcome of disease. Nos2–/– mice reproduced human-like granulomas in the lung when infected with M. tuberculosis in the ear dermis. The granulomas were hypoxic and contained large amounts of the serine protease cathepsin G and clade B serine protease inhibitors (serpins). Extrinsic inhibition of serine protease activity in vivo resulted in distorted granuloma structure, extensive hypoxia, and increased bacterial growth in this model. These data suggest that serine protease activity acts as a protective mechanism within hypoxic regions of lung granulomas and present a potential new strategy for the treatment of tuberculosis.

Authors

Stephen T. Reece, Christoph Loddenkemper, David J. Askew, Ulrike Zedler, Sandra Schommer-Leitner, Maik Stein, Fayaz Ahmad Mir, Anca Dorhoi, Hans-Joachim Mollenkopf, Gary A. Silverman, Stefan H.E. Kaufmann

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Figure 6

Serine protease activity in hypoxic regions of granulomas protects against tuberculosis.

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Serine protease activity in hypoxic regions of granulomas protects again...
(A) The bacterial burden of tissue homogenates from dermal-infected IFN-γ–blocked Nos2–/– mice in dLN, spleen, and lung after twice weekly treatment with AEBSF. CFUs at day 56 p.i. are shown as mean ± SEM (n = 5). The control group did not receive AEBSF. (B) Vmax of chymotrypsin-like serine protease activity in lung lysates at day 56 p.i. from control and AEBSF-treated mice (n = 5). AEBSF treatment resulted in a significant reduction in Vmax in the lung but not complete inhibition of chymotrypsin-like serine protease activity. Horizontal lines indicate the mean Vmax. (C) H&E staining of lung granulomas with central necrosis from control and AEBSF-treated mice (original magnification, ×100) at day 56 p.i. revealed loss of granuloma structural integrity. Acid-fast M. tuberculosis was detected using a modified basic fuchsin stain in AEBSF-treated mice, which identified enhanced bacterial growth in regions encircling the necrotic zone demonstrated to be hypoxic, demonstrating elevated Ctsg and serpinb3a/b/c expression. Regions within the rectangles are shown at higher magnification (original magnification, ×1,000). (D) PIMO staining of granulomas in control mice and AEBSF-treated mice (original magnification, ×50), for which staining shows distortion of granuloma structure and extensive development of hypoxia in AEBSF-treated mice compared with control mice. *P < 0.05.