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Published online before print February 14, 2006
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Academic Units of Respiratory Medicine and Infectious Diseases, University of Sheffield, United Kingdom
1Correspondence: Academic Unit of Respiratory Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2JF, UK. E-mail: m.k.whyte{at}sheffield.ac.uk
ABSTRACT
The innate-immune response to infection is critically dependent on the antimicrobial actions of macrophages and neutrophils. Host and pathogen have evolved strategies to regulate immune-cell antimicrobial functions via alterations in cell death. Modulation of phagocyte death by bacteria is an important pathogenic mechanism. Host benefits of phagocyte apoptosis also exist, and understanding the mechanisms and consequences of apoptosis is essential before we can devise strategies to modulate this element of the innate-immune response to the host’s benefit. This is of particular importance in an organ such as the lung, in which the balance between the need to recruit phagocytes to maintain bacterial sterility and the requirement to clear recruited cells from the alveolar units to preserve physiologic gas exchange must be finely tuned to ensure survival during bacterial infection. Apoptosis clearly plays a critical role in reconciling these physiological requirements.
Key Words: apoptosis • neutrophil • macrophage • Pseudomonas aeruginosa • Streptococcus pneumoniae
The fundamental role of programmed cell death (apoptosis) in tissue homeostasis is well established. Resolution of acute inflammation requires apoptosis of invading inflammatory cells, including neutrophils and inflammatory monocyte-macrophages. This is particularly important in the lung, where accumulation of inflammatory cells in the alveolar units compromises gas exchange. Apoptosis of recruited inflammatory cells is triggered by reduction of prosurvival factors such as bacterial components [e.g., lipopolysaccharide (LPS)] and host cytokines [e.g., granulocyte macrophage-colony stimulating factor (GM-CSF), G-CSF, interleukin (IL)-1ß] following microbial clearance or removal of other inflammatory stimuli [1 ]. Clearance of apoptotic cells ensures discrete removal of primed phagocytes, without release of factors capable of inducing tissue injury, and is required for restoration of tissue homeostasis. Apoptotic cells also have important functions in modulating the innate and adaptive immune response (reviewed in ref. [2 ]). Macrophages can discriminate between apoptotic death to restore homeostasis, down-regulating production of proinflammatory cytokines and up-regulating cytokines, which initiate remodeling [3 ], and "pathological" apoptosis or necrosis, which is proinflammatory [2 , 3 ]. It is important that apoptotic cells also serve as a source of antigen for dendritic cells, which can result in tolerization or activation of CD8+ T cell responses [2 ]. Phagocytic cells have widely differing susceptibilities to apoptosis in keeping with their diverse, physiologic functions. Neutrophils are programmed to undergo constitutive apoptosis in the absence of prosurvival stimuli in keeping with their short lifespan. Monocytes are also relatively susceptible to apoptosis, but as they differentiate into resident tissue macrophages, susceptibility to apoptosis is decreased in keeping with their long lifespan.
The physiological resolution of inflammation is subverted by a variety of pathogens. An increasing number of bacteria have been shown to manipulate phagocyte lifespan [4 ]. Pathogen strategies, which regulate cell death, include pathogen-driven apoptosis as an immune-evasion strategy and inhibition of host cell apoptosis, the latter, often to permit intracellular persistence of pathogens. Less well-appreciated has been that the host response to certain bacterial infections might include macrophage apoptosis as a mechanism to down-regulate the inflammatory response and enhance bacterial clearance from the lung.
This review focuses on phagocytes, although lymphocyte apoptosis is also important in resolution of the inflammatory response in the lung, and indeed, lymphocyte apoptosis is also subject to manipulation by microorganisms. We describe two distinct models of phagocyte apoptosis during bacterial infection in the lung with opposing effects on outcome of the host-pathogen interaction.
A large number of microbial virulence factors have been shown to increase macrophage apoptosis and thus, impair antibacterial defenses [4 ]. The classic example is Shigella flexneri infection, where the invasin ipaB directly activates caspase-1, inducing apoptosis and inflammation via cytokine release [5 ]. This form of apoptosis occurs early after exposure of macrophages to pathogens and facilitates immune evasion. It is a feature of a number of bacterial infections, particularly enteric infections caused by organisms such as Shigella sp, Salmonella sp, and Yersinia sp. A variety of pulmonary pathogens also induces macrophage apoptosis, including Legionella pneumophilia, Chlamydia psittaci, and Coxiella burnetii [4 ]. However, evidence that macrophage apoptosis benefits the host response during bacterial infection has been difficult to obtain. In macrophages infected with Mycobacterium tuberculosis, a chronic, intracellular pathogen, apoptosis limits intracellular replication, and conversely, intracellular replication is prolonged by inhibition of apoptosis. Indeed, virulence of M. tuberculosis strains correlates with ability of the pathogen to suppress apoptosis of infected cells [6 ]. Interpretation of this model is, however, complicated by the chronic nature of infection, making it difficult to determine the relative contribution of host and pathogen to the resultant apoptosis. The principle that macrophage apoptosis might benefit the host is supported by the observation that many bacteria have evolved mechanisms to facilitate survival within macrophages. For example, L. pneumophila prolongs macrophage survival during infection by up-regulation of the Naip inhibitor of apoptosis [7 ].
We have characterized macrophage apoptosis in response to infection with Streptococcus pneumoniae (the pneumococcus), the most frequent cause of community-acquired, bacterial pneumonia [8 ]. Pneumococcal pneumonia is the classic example of a bacterial infection in which the host response has been optimized to induce rapid recruitment of phagocytes to clear infection but with subsequent, complete resolution of the inflammatory infiltrate without chronic sequelae after bacteria have been cleared [1 ]. Nonetheless, significant mortality occurs with this infection, particularly at the extremes of age, which has not changed dramatically in the antibiotic era [9 ]. Thus, innate host responses are critical to the outcome in this infection and are suboptimal in a group of infected individuals. Many deaths occur early following infection, suggesting that early skewing of the innate response has a profound impact on outcome. In addition, late deaths often occur despite effective bacterial clearance, suggesting a dysregulated inflammatory response [10 ]. However, pneumococcal pneumonia is an infrequent event in comparison with the high rates of colonization, and as microaspiration of pharyngeal contents is common, innate host defenses are finely tuned and effective at clearing small numbers of aspirated bacteria in most cases. The contribution of macrophages to innate immunity to pneumococci is suggested by the increased severity of pneumococcal disease in welders—individuals who are exposed through their occupation to inhaled macrophage toxins [11 ]—and an increased frequency of pneumococcal disease in children with defects of Toll-like receptor (TLR), signaling that significantly impairs macrophage cytokine responses to microorganisms [12 ]. We have found that alveolar macrophages are particularly important for the clearance of small numbers of bacteria in a murine model of subclinical infection [13 ].
We have shown that macrophage apoptosis is a prominent feature of pneumococcal infection in vitro and in vivo [13 , 14 ]. Analysis in vitro has shown that levels of apoptosis are enhanced by macrophage differentiation to a mature tissue phenotype [14 ]. Furthermore, apoptosis requires bacterial phagocytosis and is linked to intracellular bacterial killing [14 ]. The link to intracellular killing is explained partly by a role for nitric oxide (NO) generation in triggering a mitochondrial pathway of apoptosis in macrophages during intracellular killing [15 ] (Fig. 1 ). In contrast to many examples of macrophage apoptosis associated with pathogen evasion of the immune response [4 ], the apoptosis associated with pneumococcal killing is delayed [15 ]. The precise timing of apoptosis is regulated by expression of an antiapoptotic Bcl-2 protein, Mcl-1 [16 ], which has a short half-life and has a key role in determining macrophage viability [17 ] and so, is well-positioned to respond to changing stresses within the macrophage during killing of bacteria. We found pneumococci are eliminated effectively by a macrophage response that has two phases. An initial period of macrophage viability is assocociated with increasing nitrosative stress and intracellular bacterial killing, and during this phase, Mcl-1 up-regulation maintains macrophage survival. This phase is followed 14–16 h later by apoptosis induction and clearance of bacteria, with down-regulation of Mcl-1 and up-regulation of a BH3-only proapoptotic isoform Mcl-1Exon-1, which is generated by differential splicing of the gene [16 ]. In this model, macrophage apoptosis benefits the host. Macrophage apoptosis is enhanced by features associated with a beneficial host response to pneumococci, such as bacterial opsonization, and impeded by certain virulence factors, such as by the pneumococcal polysaccharide capsule [14 ]. In addition, specific alterations in Mcl-1 gene splicing to generate a proapoptotic isoform suggest apoptosis is host-mediated and not pathogen-driven. Inhibition of microbial killing by impairing NO production inhibits apoptosis and switches the death process to necrosis [15 ]. In vivo, the beneficial role of macrophage apoptosis is particularly apparent following challenge with low-dose inocula of pneumococci, levels that are normally cleared effectively in a macrophage-dependent manner [13 ]. In these models, caspase inhibition or overexpression of Mcl-1 in mice which contain a human Mcl-1 transgene, inhibits bacterial clearance from the lung and increases susceptibility to bacteremia [14 , 15 ]. Thus, multiple strands of evidence demonstrate that host benefit can derive from macrophage apoptosis in pneumococcal pneumonia.
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Chronic infection with P. aeruginosa is a major cause of pulmonary damage and mortality in patients with cystic fibrosis, and acute infection is observed in the immunocompromised host and in patients with ventilator-associated pneumonia. The organism has evolved a number of immunoevasive strategies [26 ], including secretion of factors that impair neutrophil phagocytosis and activation and also, type III secretion system-dependent cytotoxicity [27 ]. P. aeruginosa also generates highly diffusible, toxic, secondary metabolites, known as phenazines, and is the only organism to produce a specific phenazine, named pyocyanin [28 ].
We have shown that pyocyanin, at concentrations detected in sputum of cystic fibrosis patients [29 ], induces a seven- to tenfold acceleration of neutrophil apoptosis in vitro. Apoptosis occurs rapidly (increases from baseline are noted from 2 h after exposure) and is concentration-dependent. The exquisite sensitivity of neutrophils to apoptosis following this stimulus is the subject of ongoing study. However, pyocyanin-induced apoptosis is associated with production of reactive oxygen intermediates (ROI), which may have a specific role in induction of neutrophil apoptosis [1 ], and a central role for oxidative stress in pyocyanin-induced apoptosis was identified in the nematode, Caenorhabditis elegans [30 ]. Recent work by Ran et al. [31 ], in which a yeast library was screened for mutants with altered pyocyanin susceptibility, isolated a number of mutations in the vacuolar ATPase, an enzyme that is inactivated by ROI, further identifying a possible mechanism of pyocyanin-induced cell death.
We established a murine model of acute P. aeruginosa pneumonia to test the hypothesis that pyocyanin production is a critical determinant of outcome of P. aeruginosa infection of the lung, comparing infection with wild-type P. aeruginosa with a range of pyocyanin-deficient strains [32 ]. We demonstrated the ability of pyocyanin to impair neutrophilic host defenses by a number of distinct but possibly inter-related mechanisms. Pyocyanin production leads to reduced chemokine (macrophage inflammatory protein 2, keratinocyte-derived chemokine) and cytokine (IL-1ß, IL-6) production. There were neutrophil numbers greatly reduced in the lung after the first 24 h of infection, a consequence of reduced recruitment and of accelerated apoptosis. It is important that this was associated with impaired bacterial clearance, confirming a key role for neutrophils in effective clearance of P. aeruginosa and demonstrating that pyocyanin impairs neutrophilic host defenses, at least in part, by acceleration of neutrophil apoptosis.
Studies by Vandivier et al. [33 ] have demonstrated excessive numbers of apoptotic neutrophils in the lungs of cystic fibrosis patients. Moreover, release of elastase from necrotic neutrophils results in decreased clearance of apoptotic cells [33 ]. The overwhelming majority of these patients is infected with P. aeruginosa, and their sputum contains pyocyanin at concentrations that accelerate neutrophil apoptosis [29 ]. In addition, recent work by Watt and colleagues [34 ] suggests increased proportions of late apoptotic neutrophils and reduced, viable neutrophils in sputum from individuals infected with P. aeruginosa or Burkholderia cenocepacia, compared with those with other Gram-negative bacterial infections. We therefore speculate that the combination of pathogen-driven neutrophil apoptosis and impaired apoptotic cell clearance may underlie the neutrophil-mediated tissue injury, which contributes significantly to lung destruction in cystic fibrosis.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge funding support from the Wellcome Trust for their studies. D. H. D. is a Wellcome Senior Clinical Fellow.
Received October 3, 2005; revised December 23, 2005; accepted December 28, 2005.
REFERENCES
receptors correlates with intracellular bacterial load J. Infect. Dis. 188,1119-1131[CrossRef][Medline]
B, Bad or caspase activation J. Exp. Med. 194,113-126This article has been cited by other articles:
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  Y. Zhang, X. Li, A. Carpinteiro, and E. Gulbins Acid Sphingomyelinase Amplifies Redox Signaling in Pseudomonas aeruginosa-Induced Macrophage Apoptosis J. Immunol., September 15, 2008; 181(6): 4247 - 4254. [Abstract] [Full Text] [PDF]
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L. R. Prince, S. M. Bianchi, K. M. Vaughan, M. A. Bewley, H. M. Marriott, S. R. Walmsley, G. W. Taylor, D. J. Buttle, I. Sabroe, D. H. Dockrell, et al. Subversion of a Lysosomal Pathway Regulating Neutrophil Apoptosis by a Major Bacterial Toxin, Pyocyanin J. Immunol., March 1, 2008; 180(5): 3502 - 3511. [Abstract] [Full Text] [PDF]
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