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 4

Exogenous CTSG enhances cell death of IFN-γ–activated M. tuberculosis–infected macrophages, while controlling intracellular M. tuberculosis growth in vitro when NO production is abrogated.

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Exogenous CTSG enhances cell death of IFN-γ–activated M. tuberculosis–in...
(A) Lung tissue sections derived from individuals diagnosed with active pulmonary tuberculosis (n = 6). Macrophages and granulocytes expressing CTSG, as detected by immunohistochemical staining (original magnification, ×200), localized to the central necrotic region (n = 3). Expression of CTSG was not evident within end-stage cavities in human tissues, indicating expression is consistent with active caseation in human granulomas (n = 3). (B) Level of necrosis of IFN-γ–activated WT or Nos2–/– BMDMs, cultured in vitro in the presence of exogenous CTSG. Cell death was measured as the percentage lactase dehydrogenase (LDH) activity in cell supernatants at 72 hours p.i. (MOI 0 or 2). NO production was abrogated in activated macrophages by addition of the competitive inhibitor of NOS2 l-NMMA, with the nonactive isomer d-NMMA as a control. Percentage LDH activity is shown as mean ± SD (n = 6). (C) Percentage LDH activity in cell supernatants and (D) [3H] uracil incorporation (MOI 2) of IFN-γ–activated Nos2–/– BMDMs, cultured in vitro in the presence of exogenous CTSG or NE (mean ± SD; n = 4). *P < 0.05; **P < 0.01.