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
View: Text | PDF
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

×

Figure 5

Clade B serpins are expressed in hypoxic regions of caseating granulomas, and endogenous Serpinb3a limits death of activated M. tuberculosis–infected macrophages.

Options: View larger image (or click on image) Download as PowerPoint
Clade B serpins are expressed in hypoxic regions of caseating granulomas...
(A) Staining of lungs from dermal-infected WT (normal lung), Nos2–/– (nonnecrotizing granulomas), and IFN-γ–depleted Nos2–/– (caseous granuloma) mice with anti-Serpinb3a mAbs at day 56 p.i. (original magnification, ×200). serpinb3a/b/c was detected in regions encircling the central necrotic region of caseous granulomas in IFN-γ–depleted Nos2–/– mice. (B) Relative mRNA expression of serpinb3a/b/c in individual 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. Average relative mRNA expression is shown as mean ± SEM (n = 5). (C) Expression of Ctsg and serpinb3a/b/c from IFN-γ–activated BMDMs infected with M. tuberculosis for 4, 24, and 72 hours, measured by qRT-PCR on transcribed cDNA relative to uninfected IFN-γ–activated BMDMs, with expression levels normalized to expression of GAPDH. Data are shown as mean ± SEM of 4 separate experiments. (D) Percentage of cell death of WT or Serpinb3a–/– BMDMs cultured in vitro, with or without IFN-γ–activation, measured as the percentage LDH activity in cell supernatants at 72 hours p.i. (MOI 2). Percentage LDH activity is shown as mean ± SD (n = 6). M. tuberculosis growth, evaluated by [3H] uracil incorporation at 72 hours p.i., is shown as mean cpm ± SD (n = 6). **P < 0.01.