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Nitric oxide levels regulate macrophage commitment to apoptosis or necrosis during pneumococcal infection

HELEN M. MARRIOTT^*, FARZANA ALI^*, ROBERT C. READ^*, TIM J. MITCHELL, MOIRA K. B. WHYTE^* and DAVID H. DOCKRELL^*,1

^* Division of Genomic Medicine, University of Sheffield School of Medicine and Biomedical Sciences, Sheffield, UK; and
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK

¹Correspondence: Division of Genomic Medicine, F-Floor, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK. E-mail: d.h.dockrell{at}sheffield.ac.uk

SPECIFIC AIMS

To determine whether nitric oxide (NO) contributes to host-mediated macrophage apoptosis during infection with the gram-positive bacteria Streptococcus pneumoniae (the pneumococcus) and to examine host and microbial factors that regulate this process.

PRINCIPAL FINDINGS

1. Human macrophages up-regulate inducible nitric oxide synthase (iNOS) and produce increased levels of NO after infection with pneumococci
We have examined whether human macrophages increase production of NO during pneumococcal infection using the model of in vitro infection of human monocyte-derived macrophages (MDM) with opsonized pneumococci. Direct evidence of increased NO production after pneumococcal infection compared with mock infection was provided by DAF-FM staining using flow cytometry and confocal microscopy, while indirect evidence of increased NO production was provided by increased levels of nitrite in extracellular supernatants and by increased detection of nitrotyrosine (a reactive nitrogen species; RNS) by confocal microscopy. iNOS protein levels were up-regulated after pneumococcal infection, and iNOS inhibition decreased NO production by all techniques.

2. NO contributes to intracellular killing of pneumococci
NO was produced in microvesicles adjacent to but distinct from lysosomes and pneumococci. NO accumulated with time after pneumococcal infection. iNOS inhibition significantly decreased intracellular killing of pneumococci 6 and 16 h after infection (Fig. 1 ).

View larger version (11K): [in this window] [in a new window]   Figure 1. NO contributes to killing of intracellular pneumococci. A) The number of bacteria internalized by monocyte-derived macrophages (MDM) 4 h postinfection with type 1 pneumococci in the absence (–) or presence (+) of the iNOS inhibitor 1400W. Results represent the mean and SE obtained from 4 donors. B) Intracellular killing assays were performed in MDM 6 and 16 h postinfection with pneumococci. Results represent mean and SE obtained from the same 4 donors as in panel A; *P < 0.05, Spn+ vs. Spn+ 1400W+, Wilcoxon signed rank test.

3. NO plays a role in pneumococcal-associated macrophage apoptosis
We have previously demonstrated that macrophage apoptosis during pneumococcal infection is associated with phagocytosis and killing of bacteria. iNOS inhibition was found to decrease the level of detectable apoptosis after phagocytosis of opsonized pneumococci. The decrease in apoptosis was confirmed by demonstrating both DNA stand breaks and condensed or fragmented nuclei (Fig. 2 ). NO alone was insufficient to induce apoptosis; the NO donor NOC-18 failed to induce a significant increase in macrophage apoptosis vs. control treatment in mock-infected macrophages. The combination of pneumococcal infection and NO donor, however, increased macrophage apoptosis.

View larger version (19K): [in this window] [in a new window]   Figure 2. NO production is associated with apoptosis induction in macrophages exposed to pneumococci. A) Monocyte-derived macrophages (MDM) were mock-infected (Spn-) or infected with type 1 pneumococci (Spn+) for 20 h in the presence (+) or absence (–) of 1400W and apoptosis detected by TUNEL with propidium iodide counterstaining. Images were recorded by fluorescence microscopy; green/yellow cells are TUNEL+. B) The % TUNEL+ cells were quantified by fluorescence microscopy. Results represent the mean and SE of % apoptosis for duplicate samples obtained from 5 donors. **P < 0.01 Spn+ vs. Spn+ 1400W+ Mann-Whitney U test. C) Nuclear features of apoptosis recorded in MDM 20 h postinfection in the presence (+) or absence (–) of 1400W or L-NMA. Results represent the mean and SE of % apoptosis for duplicate samples obtained from 4 donors, *P < 0.05 Spn– vs. Spn+, Spn+ vs. Spn+ 1400W+ and Spn+ vs. Spn+ L-NMA+, Wilcoxon signed rank test. D) Nuclear features of apoptosis recorded in MDM 20 h postinfection in the presence or absence of the indicated concentrations of NOC-18. Results represent the mean and SE of % apoptosis obtained from duplicate samples from 3 donors, *P < 0.05 Spn+ vs. Spn+ 100 µg/mL NOC-18+, Kruskal-Wallis test with Dunn’s Multiple Comparison.

4. NO-mediated macrophage apoptosis during pneumococcal infection involves mitochondrial membrane permeabilization and in the presence of iNOS inhibition pneumococcal infection results in macrophage necrosis
Macrophage apoptosis in association with pneumococcal infection was found to be associated with two features of mitochondrial membrane permeabilization (MMP): loss of the mitochondrial inner transmembrane potential (_m) and translocation of cytochrome c to the cytosol. iNOS inhibition prevented development of MMP. Pneumococcal-associated macrophage apoptosis was also associated with down-regulation of a key regulator of macrophage survival, the anti-apoptotic Bcl-2 family member myeloid cell leukemia sequence (Mcl) -1; iNOS inhibition reversed this change. Inhibition of the adenine nucleotide translocator with bongkrekic acid, a known inhibitor of Bax- and Bid-mediated MMP, significantly reduced pneumococcal-associated macrophage apoptosis. This suggested that MMP was a central event in pneumococcal-associated macrophage apoptosis. When pneumococcal infected macrophages were treated with an iNOS inhibitor, cell numbers decreased relative to numbers in mock-infected cultures. This also occurred after pneumococcal infection in the absence of iNOS inhibition, suggesting that cell viability was not preserved by iNOS inhibition. Cells with activated caspases in the absence of iNOS inhibition were changed to cells demonstrating loss of membrane integrity after iNOS inhibition, consistent with development of necrosis after iNOS inhibition.

5. Pneumolysin contributes to NO-mediated macrophage apoptosis during pneumococcal infection
Purified pneumolysin induced significant macrophage apoptosis, but at much lower levels than seen with internalized whole bacteria. The importance of pneumolysin was confirmed as a pneumolysin-deficient strain of pneumococcus induced less NO and less apoptosis than its parental pneumolysin-sufficient strain.

CONCLUSIONS AND SIGNIFICANCE

1. NO is produced in human macrophages during pneumococcal infection
Although best characterized in rodent macrophages, many studies have demonstrated NO production in human macrophages. We have extended these findings by demonstrating that pneumococci stimulate NO production by human macrophages. Although the levels produced in human macrophages may be lower than in rodent cells, we document important functions for NO in host defense against pneumococci, including evidence for a role in intracellular killing. Although established for chronic intracellular infection of human macrophages, these important functions of NO have not been previously identified during acute resolving bacterial infections as occurs with pneumococcal infection.

2. NO-mediated macrophage apoptosis during pneumococcal infection
NO has been demonstrated to both enhance and inhibit macrophage apoptosis depending on experimental conditions. These findings were largely based on the use of NO donors. In mycobacterial infection, some studies (but not others) have suggested NO contributes to macrophage apoptosis, but interpretation of these findings is complicated by the chronic nature of mycobacterial infection. Apoptosis of MDM after in vitro exposure to pneumococci is a delayed response and does not occur during the initial period of NO production associated with intracellular killing. At later times after infection, we observed that NO played a critical role in determining the fate of macrophages and induced MMP-dependent apoptosis.

Our study extends earlier observations by studying the effect of NO on macrophage apoptosis in phagocytosis and intracellular killing of bacteria. Under these conditions we confirm that as NO accumulates, NO induces MMP. In comparison to some studies involving NO donors, macrophage apoptosis in our model is associated with loss of _m and MMP is a central event in apoptosis induction. An explanation for these differences may be that additional signals contribute to apoptosis during pneumococcal infection of macrophages, as supported by the finding that NO is necessary but not sufficient for macrophage apoptosis in this model. The additional signals may be provided in response to pneumococcal factors and may act by NO-independent signaling pathways. Pneumococcal pneumolysin has been implicated in neuronal apoptosis, but this response occurs efficiently with exogenous pneumolysin and is different from macrophage apoptosis, which requires internalized bacteria.

In the presence of iNOS inhibition, macrophages that have internalized pneumococci become susceptible to a necrotic cell death. Induction of necrosis in the presence of iNOS inhibition is likely the result of ATP depletion in association with a decrease in cytosolic pH, a feature of phagocytes that contain large numbers of intracellular bacteria. The level of NO therefore has an important role in determining the mechanism of cell death during pneumococcal infection of macrophages.

3. Potential significance of host-mediated macrophage apoptosis induced by NO
NO can, under certain circumstances, decrease the development of inflammation. Induction of macrophage apoptosis represents another example of how NO may regulate resolution of inflammation after infection by removing activated cells and limiting tissue damage. Macrophage apoptosis provides a source of antigens for presentation by dendritic cells, hence linking innate and adaptive immunity. Host-mediated macrophage apoptosis may also have a role in microbial killing. The importance of macrophage apoptosis in controlling microbial replication is best characterized for mycobacterial infection, but interpretation is complicated by the chronic nature of infection. In contrast, pneumococcal infection of macrophages is acute and phagocytosed bacteria are completely cleared. Pneumococci, unlike mycobacteria, have not been demonstrated to possess genes conferring resistance to nitrosative stress and pneumococcal infection may be a more effective example of NO-induced host-mediated macrophage apoptosis.

CONCLUSIONS

We provide evidence of the expanding role of NO in host defense against bacteria in human macrophages. At lower concentrations, NO contributes to pneumococcal killing; at higher concentrations it facilitates MMP-mediated apoptosis. These findings implicate NO as an important factor in host-mediated macrophage apoptosis during pneumococcal infection and support its importance in resolution of inflammation during infection.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1450fje; doi: 10.1096/fj.03-1450fje

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