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 3

Hypoxia and Ctsg expression is associated with granuloma pathology.

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Hypoxia and Ctsg expression is associated with granuloma pathology. (A) ...
(A) Dermal-infected mice (day 56 p.i.), with and without blocking of TNF-α or IFN-γ, received the tissue hypoxia marker PIMO (n = 5) and were sacrificed 2 hours later, and lung sections were stained for the presence of PIMO adducts. One hundred percent of caseous necrotic granulomas in all groups stained positive for PIMO adducts. Images show typical PIMO staining in granulomas after cytokine blocking (original magnification, ×100 [top]; ×200 [bottom]). (B) Numbers of PIMO-positive granulomas (PIMO +ve) were significantly higher after blocking of TNF-α or IFN-γ (mean ± SEM; n = 5). (C) Relative mRNA expression of Ctsg in individual Nos2–/– versus WT mice and IFN-γ–depleted Nos2–/– versus untreated Nos2–/– mice at day 28 p.i., measured by qRT-PCR on transcribed cDNA and normalized to expression of GAPDH (mean ± SEM; n = 5). (D) Staining of lung tissue from dermal-infected WT (normal lung), Nos2–/– (nonnecrotizing), and IFN-γ–depleted Nos2–/– (caseous granuloma) mice with anti-Ctsg mAbs at day 56 p.i. (original magnification, ×200). Staining shows cells within nonnecrotizing granulomas expressing Ctsg in Nos2–/– mice. Ctsg was detected in regions encircling the central necrotic regions of caseous granulomas in IFN-γ–depleted Nos2–/– mice that were identified as hypoxic (Figure 3A). *P < 0.05; **P < 0.01.