Macrophage elastase kills bacteria within murine macrophages - PubMed (original) (raw)

. 2009 Jul 30;460(7255):637-41.

doi: 10.1038/nature08181. Epub 2009 Jun 17.

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Macrophage elastase kills bacteria within murine macrophages

A McGarry Houghton et al. Nature. 2009.

Abstract

Macrophages are aptly positioned to function as the primary line of defence against invading pathogens in many organs, including the lung and peritoneum. Their ability to phagocytose and clear microorganisms has been well documented. Macrophages possess several substances with which they can kill bacteria, including reactive oxygen species, nitric oxide, and antimicrobial proteins. We proposed that macrophage-derived proteinases may contribute to the antimicrobial properties of macrophages. Macrophage elastase (also known as matrix metalloproteinase 12 or MMP12) is an enzyme predominantly expressed in mature tissue macrophages and is implicated in several disease processes, including emphysema. Physiological functions for MMP12 have not been described. Here we show that Mmp12(-/-) mice exhibit impaired bacterial clearance and increased mortality when challenged with both gram-negative and gram-positive bacteria at macrophage-rich portals of entry, such as the peritoneum and lung. Intracellular stores of MMP12 are mobilized to macrophage phagolysosomes after the ingestion of bacterial pathogens. Once inside phagolysosomes, MMP12 adheres to bacterial cell walls where it disrupts cellular membranes resulting in bacterial death. The antimicrobial properties of MMP12 do not reside within its catalytic domain, but rather within the carboxy-terminal domain. This domain contains a unique four amino acid sequence on an exposed beta loop of the protein that is required for the observed antimicrobial activity. The present study represents, to our knowledge, the first report of direct antimicrobial activity by a matrix metallopeptidase, and describes a new antimicrobial peptide that is sequentially and structurally unique in nature.

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Figures

Figure 1

Figure 1. MMP12 provides survival advantage in Gram-positive and Gram-negative infection

a, Kaplan–Meier curve for _Mmp12_−/− and wild-type (WT) mice after i.p. injection with 1 ×108 c.f.u. S. aureus (n = 40 for each group from six experiments); P = 0.0014, log-rank test. b, c, Kaplan–Meier curves for low (b; 1 ×107 c.f.u.) and high (c; 1 ×109 c.f.u.) S. aureus titres (n = 6 each group); _P <_0.05. **d**, Bacterial c.f.u. 24 h after i.p. _S. aureus_ injection in peritoneal fluid (PF), blood, liver and lungs (_n_ = 4 each group); *_P <_0.01. **e**, **f**, Peritoneal macrophage (macs; **e**) and neutrophil (**f**) counts 2 and 24 h after i.p. _S. aureus_ injection (_n_ = 6 each group). PMN, polymorphonuclear leukocytes; _P >_0.05. g, Kaplan–Meier curve for _Mmp12_−/− (n = 15) and wild-type (n = 16) mice (from three experiments) after 1 ×108 c.f.u. i.p. E. coli injection; P = 0.0271. h, Kaplan–Meier curve for intravenous S. aureus (1 ×108 c.f.u.) to wild-type (n = 13) and _Mmp12_−/− (n = 16) mice (from three experiments); P = 0.9640. i, Lung bacterial burden (c.f.u.) 2 h after S. aureus TVI (n = 6 each group); *_P <_0.005. All error bars represent s.d.

Figure 2

Figure 2. MMP12 improves bacterial clearance and survival in S. aureus pneumonia

a, Kaplan–Meier curve for Mmp12+/+(n = 30; wild-type, WT) and _Mmp12_−/− (n = 36) mice (from six experiments) administered with S. aureus i.t. (1 ×108 c.f.u.); P = 0.0093, log-rank test. b, Bacterial burden in the lung after sub-lethal dose of S. aureus i.t. (1 ×106 c.f.u.; n = 8 each group); *_P<_0.01. c, Intramacrophage bacterial particle counts on Gram stain (n =8 each group); *_P <_0.001. d, Mmp12+/+ and _Mmp12_−/− alveolar macrophages on Gram stain at the 2-h time point. Original magnification, ×100. All error bars represent s.d.

Figure 3

Figure 3. MMP12 is required for early intracellular bacterial killing by macrophages

Peritoneal macrophages were incubated with S. aureus in RPMI plus 10% FCS without antibiotics for 60 min (t = 0). a, Intracellular bacterial c.f.u. from macrophage lysates. Data are from an experiment in triplicate. Error bars represent s.d., *_P <_0.05. b, Scanning electron microscopy (original magnification, ×4,400) of macrophages (from three experiments) at t = 60 min. c, Staphylococcus aureus in phagolysosomes of wild-type macrophages. Immuno-electron-microscopy (original magnification, ×30,000) shows MMP12 (gold particles) on S. aureus cocci (c, d) within wild-type macrophages. Gold particles were not detected within _Mmp12_−/− macrophages (d).

Figure 4

Figure 4. MMP12 CTD possesses bactericidal activity

a–c, Bacteria were incubated with human pro-MMP12 in 5% TSB (a) or RPMI plus 10% FCS (b), or with mouse MMP12 CTD (c) for 60 min at 37 °C. Data are expressed as c.f.u. (from three experiments); *_P <_0.005. d, Fluorescent propidium iodide exclusion assay after co-incubation of S. aureus and MMP12 CTD or control for 30 min. All bacteria stain positive for Syto 59 (red), but only those with disrupted membranes stain with S-7020 (green). e, Human pro-MMP12 incubated with and without S. aureus to study CTD processing in the presence of bacteria using a CTD antibody. The 54 kDa band represents full-length MMP12, the 45 kDa band represents shedding of pro-domain, and the 25 kDa band represents the CTD. The CTD is not processed to smaller fragments. f, Computational three-dimensional model of mouse MMP12 CTD. The SR-20 sequence is located within CTD blade II including β strands β2 and β3, as well as the connecting and flanking loops (green trace). g, SR-20 shows a high degree of homology among MMP12 orthologues but not among other MMPs. h, Staphylococcus aureus was incubated with either wild-type (WT) MMP12 SR-20 peptide or mutant peptide (Lys-Asp-Glu-Lys replaced by Ser-Gly-Arg-Gln) (both at 20 μg ml−1), in RPMI plus 10% FCS for 60 min. Data are expressed as c.f.u., *_P <_0.001. All error bars represent s.d.

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