Dual Role of Hydrogen Peroxide as an Oxidant in Pneumococcal Pneumonia

Abstract

Significance: Streptococcus pneumoniae (Spn), a facultative anaerobic Gram-positive human pathogen with increasing rates of penicillin and macrolide resistance, is a major cause of lower respiratory tract infections worldwide. Pneumococci are a primary agent of severe pneumonia in children younger than 5 years and of community-acquired pneumonia in adults. A major defense mechanism toward Spn is the generation of reactive oxygen species, including hydrogen peroxide (H₂O₂), during the oxidative burst of neutrophils and macrophages. Paradoxically, Spn produces high endogenous levels of H₂O₂ as a strategy to promote colonization.

Recent Advances: Pneumococci, which express neither catalase nor common regulators of peroxide stress resistance, have developed unique mechanisms to protect themselves from H₂O₂. Spn generates high levels of H₂O₂ as a strategy to promote colonization. Production of H₂O₂ moreover constitutes an important virulence phenotype and its cellular activities overlap and complement those of other virulence factors, such as pneumolysin, in modulating host immune responses and promoting organ injury.

Critical Issues: This review examines the dual role of H₂O₂ in pneumococcal pneumonia, from the viewpoint of both the pathogen (defense mechanisms, lytic activity toward competing pathogens, and virulence) and the resulting host–response (inflammasome activation, endoplasmic reticulum stress, and damage to the alveolar–capillary barrier in the lungs).

Future Directions: An understanding of the complexity of H₂O₂-mediated host–pathogen interactions is necessary to develop novel strategies that target these processes to enhance lung function during severe pneumonia.

Introduction

Infections of the lower respiratory tract represent the main cause of infectious disease mortality and represent the fifth highest cause of death overall. In 2015, pneumonia accounted for 2.7 million deaths worldwide (47). Streptococcus pneumoniae (Spn) is a major etiologic agent of community-acquired pneumonia and the leading cause of death in children younger than 5 years, worldwide (50, 51). It is a facultative anaerobe gram-positive bacterium that can colonize the upper respiratory tract as a commensal bacterium in healthy individuals. This asymptomatic transitory phase can lead to severe pneumonia upon migration of bacteria to the lower respiratory tract (119). Infections with Spn can lead to meningitis after crossing the blood–brain barrier and the following entry into the blood stream can contribute to heart disease by causing microlesions in the myocardium (14).

Currently, two main types of vaccines have been developed to reduce or eliminate the burden of infections, that is, the unconjugated 23-valent polysaccharide vaccine and a 10- or 13-valent-conjugated polysaccharide vaccine. However, their serotype coverage is not universal and they are limited with respect to noncapsulated Spn (12, 97). Once symptoms of pneumonia are diagnosed, antibiotic therapy with amoxicillin for ages under five, and azithromycin or clarithromycin for ages over five is indicated (38, 71). Antibiotic therapy, however, needs to be initiated before breakdown of the alveolar–capillary barrier and systemic inflammation in pneumococcal pneumonia (8). Unfortunately, in view of emerging pathogens, demographic shifts toward older populations, and increasing antibiotic resistance, the occurrence of pneumonia is poised to worsen rather than improve in the coming years (30, 119).

Significance

Bacterial pneumonia represents one of the major comorbidities that occur with the acute respiratory distress syndrome (ARDS) (23, 48, 81). One of the major lethal complications of ARDS is the development of pulmonary edema. Indeed, pathological specimens from ARDS patients reveal diffuse alveolar damage, and animal studies of bacterial pneumonia-associated ARDS have demonstrated both alveolar epithelial and lung endothelial injury with accumulation of protein-rich fluid in the alveolar space (27, 55, 78, 88). The ability of Spn to promote lung disease in the human host depends not only on microbial virulence factors, such as the pore-forming toxin pneumolysin (Ply) (77, 79, 111, 137), but also on variables in the host (age, genetic, and environmental factors). These affect the capacity of the immune system to clear bacteria and the susceptibility to develop tissue damage (30, 61).

While there has been focus on the pathogens causing pneumonia in recent years, for example, on Spn, there is a high and urgent need for research from the perspective of the host. The absence of alveolar neutrophilia was shown to be deleterious in murine pneumonia models (8, 101) and has a high negative predictive value for bacterial pneumonia in critically ill patients with suspected infection (129). Reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), play an important but highly complex role in pneumococcal pneumonia-associated ARDS. On the one hand, they are involved in neutrophil- and alveolar macrophage-mediated antibacterial defense, but on the other hand, they can contribute to the dysfunction of the alveolar–endothelial barrier and impair alveolar liquid clearance mechanisms. The following chapters discuss the actions of H₂O₂ from the viewpoint of the pathogen as well as in the host and outline its roles in driving the unfolded protein response (UPR), in blunting immune defense mechanisms, and in promoting acute lung injury.

Recent Advances: Actions of H₂O₂ on Spn

Spn generates and exploits H₂O₂

Spn, which grows predominantly in an aerobic environment of the oxygen-rich airway surface, can endogenously generate millimolar levels of H₂O₂ as a by-product during aerobic growth (90, 104). Here we describe those enzymes involved in pneumococcal H₂O₂ generation and examine how the pathogen exploits these high H₂O₂ levels to promote its colonization and enhance its virulence.

Enzymes involved in H₂O₂ generation in Spn

Pyruvate oxidase (SpxB) is considered the main source of H₂O₂ in Spn, as strains lacking this enzyme produce 87% less H₂O₂ (74). SpxB catalyzes the conversion of pyruvate to the phosphoryl donor, acetyl phosphate, and releases CO₂ and H₂O₂ as by-products (104). Lactate oxidase (LctO), which converts lactate to pyruvate, positively impacts pyruvate flux through SpxB. Spn mutants lacking lactate oxidase also produce significantly lower levels of H₂O₂ (38% of the total H₂O₂) (74, 90, 104, 122) (Overview in Table 1).

Table 1.

Overview of Pneumococcal Enzymes Involved in the Generation of and Protection from Hydrogen Peroxide

Deletion of spxB and lctO significantly reduces bacterial H₂O₂ production. The absence of these enzymes renders Spn highly susceptible to the presence of exogenous H₂O₂ stress generated by host phagocytic cells to facilitate clearance of bacteria at infection sites (74, 104). This suggests a link between endogenous bacterial H₂O₂ generation and resistance to exogenous H₂O₂ produced by cells of the immune system. Levels of adenosine triphosphate (ATP) decreased more rapidly in ΔspxB pneumococcal mutants than in the wild-type strain during H₂O₂ exposure, suggesting that the increased killing of these mutants is due to their inability to limit depletion of ATP during H₂O₂ stress (104).

High levels of secreted H₂O₂ promote Spn colonization

Endogenously generated H₂O₂ diffuses rapidly through cell membranes and accumulates in the extracellular milieu of Spn cultures in concentrations high enough to kill or inhibit other common inhabitants of the respiratory tract, such as Haemophilus influenzae, Staphylococcus aureus, Neisseria meningitidis, and Moraxella catarrhalis (103). Using an in vitro model mimicking Spn/S. aureus contact during colonization of the nasopharynx, pneumococcal SpxB/LctO-produced H₂O₂ was shown to be converted into the more potent oxidant hydroxyl radical (^•OH) that rapidly kills S. aureus (139). Thus, high levels of H₂O₂ provide a competitive advantage to Spn over other pathogens and commensal bacteria. A similar strategy is also applied by lactic acid bacteria at other epithelial interfaces, such as in the gut or the vagina. Thus, excretion of high levels of H₂O₂ by vaginal lactobacilli (e.g., L. gasseri, L. acidophilus, and L. johnsonii) represents one of the most important defense mechanisms against vaginal colonization by undesirable microorganisms (58, 85).

Adaptation to endogenously generated H₂O₂ in Spn

Lisher et al. (74) revealed that SpxB/LctO-generated H₂O₂ functions as an intracellular signaling molecule in Spn that modulates glycolytic-, nucleotide-, and capsule-biosynthesis via protein sulfenylation. The authors demonstrated a clear correlation between sulfenylation levels and endogenous H₂O₂ production, with SpxB and LctO being among the identified targets of protein sulfenylation. Sulfenylation of SpxB was proposed to allow it to function as an H₂O₂ “sink,” which is consistent with the fact that ΔspxB strains are more sensitive to exogenous H₂O₂ (74). Similarly, the thiol peroxidase (TpxD), which limits sulfenylation, was also shown to play a critical role in the adaptation to endogenous H₂O₂ in Spn (74).

A serine/threonine kinase StkP phosphorylates the response regulator ComE to control different cellular processes, including H₂O₂ production (107). Deletion of comE and stkP reduced the expression of spxB and tpxD and significantly decreased H₂O₂ generation (107).

A role for pneumococcal flavin reductase in resistance to oxidative stress has also been suggested, since an inactive mutant of the enzyme significantly increased susceptibility to H₂O₂, compared with wild-type Spn. Flavin reductase activity in Spn might also to be involved in bacterial virulence, as its absence does not affect Spn phagocytosis by primary mouse peritoneal macrophages, but it blunts adhesion to the type II alveolar-like cell line A549 (94).

Endogenous H₂O₂ promotes release of the pore-forming toxin Ply

The 53-kDa cholesterol-dependent pore-forming cytolysin (cholesterol-dependent cytolysin [CDC]) Ply (77, 79, 111, 137) can be released in the lungs by autolysis or upon antibiotic-mediated lysis (2). Although Ply lacks the N-terminal signal sequence found in other CDCs, which allows for extracellular release via the Sec-dependent pathway (128), the toxin's release has also been suggested in the absence of autolysis (4). Interestingly, Spn mutants that lack the pyruvate oxidase gene (spxB) are defective in Ply release. However, whereas complementation of spxB restored Ply release, exogenous addition of H₂O₂ failed to induce it. Since catalase supplementation prevented Ply release in some strains, these results indicate that intracellularly generated rather than secreted H₂O₂ promotes Ply release in some Spn strains (17).

Another link between SpxB and Ply release has been recently described. Deoxycholate-induced autolysis of Spn was significantly reduced in an SpxB-deficient strain, indicating a possible weakening of the cell membrane when SpxB is expressed (17). Endogenous H₂O₂ was found to control membrane fatty acid composition, by specific oxidation of the active site cysteine thiol residue of FabF (7). Nevertheless, it remains to be shown as to whether H₂O₂ generated by SpxB affects the Ply release directly or rather indirectly, by affecting the expression of yet unknown pneumococcal activators or repressors.

Protective pneumococcal mechanisms against H₂O₂

Generation of ROS is a conserved strategy of host phagocytic cells, primarily neutrophils, monocytes, and macrophages, to facilitate clearance of bacteria at the infection site. Bacteria can be engulfed and enclosed in phagosomes, into which superoxide is released by activated NADPH oxidase 2 (Nox-2). The resulting superoxide O₂^−• can then be dismutated to H₂O₂ by superoxide dismutase (SOD) in macrophages, and H₂O₂ can further be converted by myeloperoxidase (MPO) in neutrophils to generate the highly microbicidal species, hypochlorous acid (64, 136). Combating endogenous and host innate immune cell-mediated oxidative stress is an integral aspect of colonization and virulence of the facultative anaerobe Spn.

Pneumococcal enzymes involved in defense from high H₂O₂ levels

Common proteins known to protect against oxidative stress in other bacterial species, such as the H₂O₂ scavengers catalase and NADPH-peroxidase, are absent in Spn (62, 123). However, previous studies have identified other pneumococcal enzymes implicated in the defense against oxidative stress (Table 1; Fig. 1), such as TpxD (53), Nox (3), SOD (143), and alkyl hydroperoxidase (alkyl hydroperoxide reductase [AhpD]) (100). The pneumococcal TpxD plays an important role in H₂O₂ elimination by catalyzing its reduction (53, 54).

FIG. 1.

Mechanisms of pneumococcal defense against H₂O₂. Schematic overview of the three defense mechanisms involved in the resistance against H₂O₂: (i) defense enzymes that directly or indirectly degrade H₂O₂, (ii) repair mechanisms, and (iii) regulators linked to H₂O₂ stress response. H₂O₂, hydrogen peroxide. Color images are available online.

Exposure to exogenous H₂O₂ (10–1000 μM) significantly upregulated the expression of tpxD in Spn, whereas deletion of tpxD decreased the growth rate and survival of pneumococci in the presence of exogenously added H₂O₂. Addition of the H₂O₂ scavenger catalase in the culture medium reversed the attenuated growth of the tpxD deletion mutant (53, 54). However, Spn lacks the transcription factors OxyR and PerR found in other bacteria such as Escherichia coli, which are described to regulate the expression of tpxD in response to H₂O₂ (18, 21, 62, 92). Instead, the transcription factor CodY was recently identified to be an activator of tpxD expression, triggering its upregulation in Spn under H₂O₂ stress (54).

The pneumococcal Nox is suggested to be indirectly involved in H₂O₂ scavenging, as it converts O₂ to H₂O and thereby prevents the generation of superoxide anion (O₂^•−) and H₂O₂, which occurs when O₂ is not fully reduced. Pneumococcal Nox is necessary for Spn virulence in the murine respiratory tract and in Mongolian gerbil otitis media infection models (146). Unexpectedly, Nox mutations are linked to changes in virulence after intraperitoneal infection, where oxygen tension is low (3). However, Nox has also been associated with other functions such as Spn adhesion to A549 epithelial cells (96).

SODs protect cells from the toxic effects of O₂^•− by converting it into the more diffusible and less reactive H₂O₂ and O₂ (73). In prokaryotes, SODs are distinguished by their metal cofactors: Fe³⁺, Mn²⁺, Cu²⁺/Zn²⁺, and Ni²⁺ (31, 37, 140). Previous studies demonstrated that E. coli mutants deficient in both the manganese- and iron-containing SODs are more sensitive to H₂O₂ (21). Furthermore, the expression of MnSOD (SodA) from S. thermophiles in bacteria not expressing SODs, such as L. gasseri and L. acidophilus, provided protection against H₂O₂ stress (16). It seems that SOD protects cells against H₂O₂ by removing O₂^•− and preventing the redox cycling of iron.

Although Spn contains two types of SODs—MnSOD and FeSOD—only MnSOD (SodA) levels were increased during aerobic growth, and deletion of SodA lowered the growth of the bacterium in aerobic conditions. Virulence studies revealed attenuated growth of the sodA deletion mutant in intranasally infected mice, with growth rates in the lung and bloodstream comparable with wild type (143). In conclusion, the mechanism by which SOD provides protection of Spn against H₂O₂ is indirectly targeted at removing O₂^•− and preventing the redox cycling of iron.

A putative gene for ahpD, a group of enzymes known to degrade H₂O₂, was detected in Spn (92, 100). However, AhpD does not seem to provide H₂O₂ resistance in Spn, as its depletion rather increased the resistance of Spn against H₂O₂ (100).

Pneumococcal proteins involved in repair of H₂O₂-induced damage

Cellular damage caused by oxidative stress is multilayered and rapid. Therefore, in addition to enzymatic degradation of H₂O₂, immediate repair of damaged proteins and lipids is essential for the survival of the bacterium. Spn is equipped with a number of proteins known to be involved in the repair of H₂O₂-induced damage. These proteins include the following: (i) high-temperature requirement A, a serine protease, and chaperone protein, (ii) Clp ATP-dependent proteases, and (iii) CTM proteins consisting of CcdA (putative cytochrome c-type biogenesis protein homologue), thioredoxin-like protein family (TlpA), and methionine sulfoxide reductase A/B protein (MsrAB). All of these proteins participate in the recovery from H₂O₂-induced oxidative stress, as their progressive removal leads to an increased susceptibility of Spn to H₂O₂ [reviewed in ref. (142)]. Table 1 and Figure 1 summarize the mode of action and the regulators of proteins involved in pneumococcal defense against H₂O₂.

Impact of H₂O₂ on iron homeostasis

Transition of Spn from the nasopharynx to the lungs requires adjustment to a variety of environmental conditions, including availability of transition metal ions (98). Iron (Fe²⁺) potentiates oxidative stress. Expression of Fe²⁺ transport systems and proteins that protect against oxidative stress is regulated by the orphan response regulator RitR. Deletion of ritR impaired Spn growth in high-Fe²⁺ medium, which could be reversed upon addition of manganese (Mn²⁺), the latter of which caused a reduction in the amount of H₂O₂ produced by Spn.

In Spn, excess levels of Zn²⁺ prevent the uptake of Mn²⁺ by irreversibly binding to the extracellular Mn²⁺-binding protein pneumococcal surface adhesin A (89, 124). As a consequence, Spn becomes hypersensitive to oxidative stress, due to a decrease in the activity of MnSOD, which plays an important role in protecting the pathogen against high exogenous H₂O₂ levels (125). It is likely that many of the enzymes involved in resistance to ROS in Spn metallate with manganese rather than with iron.

H₂O₂ can interact with ferrous ions (Fe²⁺⁾ and form a highly reactive hydroxyl radical (^•OH) through the Fenton reaction, causing DNA damage and increased toxicity to the cells. Spn contains only a small number of iron-containing proteins, in contrast to most other bacteria, such as E. coli, and thereby avoid poisoning themselves during radical attack. This is consistent with findings showing that H₂O₂-mediated killing of Spn is unaffected by iron chelators and appears to be independent of the Fenton reaction (104).

A homologue of DNA-binding protein from starved cells (Dps) and of Dps-like peroxide resistance (Dpr) is present in the pneumococcal genome. Unlike Dps, which protects DNA through direct association, Dpr was found to be an important factor for mediating resistance of oxidative stress caused by H₂O₂ through chelation of free iron, thereby inhibiting the Fenton reaction. Furthermore, a dpr mutant has a reduced ability to colonize and is more rapidly cleared from the nasopharynx in a mouse model (65). In S. suis, Dpr can bind ferrous ions as well as other divalent cations, such as Cu²⁺, Mn²⁺, and Zn²⁺ (52).

Critical Issues: Actions of H₂O₂ on the Host's Lung Cells

Spn-derived H₂O₂ induces the UPR in host cells

Endoplasmic reticulum (ER) stress, resulting from the accumulation of unfolded proteins, increased protein load, or calcium gradient dysregulation, can trigger the UPR. This fundamental stress response is used by eukaryotic cells to match demand for protein synthesis with the capability to fold proteins within the ER, to maintain cellular homeostasis (148). In lower organisms, such as Caenorhabditis elegans, the UPR was demonstrated to participate in the defense against pathogens, since loss of this pathway was shown to induce hypersensitivity to certain pore-forming toxins, but not to other toxic insults (10).

UPR signaling is mediated by three transducers that are inserted into the ER membrane: activating transcription factor 6 (ATF6), protein kinase R (PKR)-like ER kinase (PERK), and inositol-requiring enzyme 1 (IRE1). The ER-resident chaperone immunoglobulin heavy chain-binding protein (BiP) binds to the luminal domain of the ER stress sensors and keeps them in a monomeric inactive state. Accumulation of unfolded proteins leads to release of BiP from the ER stress sensors and induces its subsequent binding to the unfolded proteins (20, 24). PERK and IRE1 become activated by dimerization (or oligomerization) and subsequent phosphorylation (19, 26). Phosphorylated IRE1 activates its own endoribonuclease, which mediates splicing from X-box-binding protein 1 (XBP1) mRNA. Translation of the spliced transcript produces active XBP1 protein that translocates to the nucleus, where it induces the transcription of several ER-resident chaperones and protein-folding enzymes (57, 70).

PERK activation induces phosphorylation of serine 51 of eukaryotic translation initiation factor 2α (eIF2α). As a consequence, translation is inhibited and protein folding stress in the ER is decreased. However, PERK activation also increases the translation of the transcription factor activating transcription factor 4 (ATF4). ATF4 mRNA possesses internal ribosome entry site sequences in its 5′-untranslated regions and as such escapes translational suppression. Upon entering the nucleus, ATF4 regulates the expression of UPR target genes (20). Dissociation of BiP from ATF6 initiates its translocation to the Golgi compartment where it can be cleaved by two proteases. The activated transcription factor ATF6 then migrates to the nucleus and activates the expression of genes encoding proteins, which enhance ER capacity and function (including BiP) (138, 145).

Although UPR signaling can alleviate ER stress, it can also—when sustained—activate deleterious cell death pathways if homeostasis cannot be restored (99). Recently, activation of the UPR was even suggested to mediate vascular disease (105, 112). Indeed, the p22phox subunit, an essential component of most Noxs, was shown to be a novel target of the UPR transcription factor ATF4 under ER stress conditions, thereby increasing ROS generation from Noxs and promoting pathogenesis of cardiovascular disease. Hence, the UPR plays an important role in physiology, but can also contribute to development of cardiovascular disease. Infection with Spn can induce the UPR (6, 70, 93). This was demonstrated by the expansion of ER membranes in the H441 cell line (human lung adenocarcinoma similar to club-like lung epithelial cells) upon infection (75), a common feature observed during the UPR (106).

Secretion of Spn-derived H₂O₂ leads to activation of PERK, ATF-6, and IRE1 (Fig. 2). Dimerization and phosphorylation of activated PERK induce phosphorylation of eIF2α leading to inhibition of protein translation and ATF4 modulating expression of target genes (Fig. 2). Activated ATF-6 translocates to the Golgi, where it is cleaved by site-1 protease and site-2 protease. The processed ATF-6 enters the nucleus acting as a transcription factor of target genes. Activation of IRE1 leads to splicing of xbp1 mRNA, which acts as transcription factor of target genes. IRE1 activation can also lead to regulated Ire1-dependent decay (RIDD of mRNA) or c-Jun N-terminal kinase (JNK) signaling activation. Induction of the UPR slows down ongoing protein synthesis and increases the folding capacity of the ER.

FIG. 2.

Actions of Spn-derived H₂O₂ on the UPR in host cells. Infection with Spn induces the activation of the UPR. Secretion of Spn-derived H₂O₂ leads to activation of PERK, ATF-6, and IRE1. Dimerization and phosphorylation of activated PERK induce phosphorylation of eIF2α leading to inhibition of protein translation and ATF4 modulating expression of target genes. Activated ATF-6 translocates to the Golgi, where it is cleaved by S1P and S2P. The processed ATF-6 enters the nucleus acting as a transcription factor of target genes. Activation of IRE1 leads to splicing of xbp1 mRNA, which acts as transcription factor of target genes. IRE1 activation can also lead to RIDD of mRNA or JNK signaling activation. ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; eIF2α, eukaryotic translation initiation factor 2α; ER, endoplasmic reticulum; IRE1, inositol-requiring enzyme 1; JNK, c-Jun N-terminal kinase; PERK, protein kinase R (PKR)-like ER kinase; RIDD, regulated IRE1-dependend decay; S1P, site-1-protease; S2P, site-1-protease; Spn, Streptococcus pneumonia; UPR, unfolded protein response; xbp1, X-box-binding protein 1. Color images are available online.

Although a number of bacterial virulence factors, including lipopolysaccharide (LPS) and some pore-forming toxins, such as listeriolysin-O, were shown to induce the UPR (10, 24, 75), infections with Spn in H441 cells revealed that H₂O₂, rather than the pore-forming toxin Ply, is the main trigger of UPR, mainly by means of activating the PERK pathway (Fig. 2). The important role of H₂O₂ in the induction of the UPR during pneumococcal infection is documented by the observation that a mutant Spn strain lacking pyruvate oxidase (SpxB), which secretes a very low level of H₂O₂, does not induce UPR in H441 cells (75).

Induction of DNA damage by pneumococcal-secreted H₂O₂

Spn not only generates large amounts of H₂O₂ endogenously (34), but also releases large quantities of H₂O₂ in vitro and in vivo (42). Host cells typically trigger an antioxidant stress response during Spn infection, to prevent oxidative injury. This protective response occurs by inducing the production of the transcription factor nuclear factor erythroid 2-related factor 2, which plays a crucial role in the transcriptional activation of antioxidant enzymes and ROS scavengers. These include heme oxygenase 1, NADPH dehydrogenase, and SOD (73, 83).

At excessive concentrations, H₂O₂ can, however, induce toxic DNA double-strand breaks, which precede apoptosis, as was shown using cultures of the human A549 cell line (a model for type II pneumocytes) exposed to three serotypes of Spn. Catalase reduced the frequency of DNA damage-positive cells in these studies by 50% or more (109).

Another consequence of bacterial H₂O₂ release is an increased production of mitochondrial ROS in the host cells, as observed during infection with wild-type Spn, with a Ply-negative mutant strain (Δply), but not with a SpxB-negative Spn strain (63, 75) (Fig. 3). Spn-secreted H₂O₂ can also cause oxidative damage to mitochondrial DNA (mtDNA) and lead to the release of mtDNA into the cytoplasm, which in turn induces type I interferon expression in the A549 cell line, involving the stimulator of interferon gene signaling pathway (46) (Fig. 3).

FIG. 3.

H₂O₂-induced responses in the host cell. Spn produces large amounts of H₂O₂, which induce a plethora of host cell responses. This includes oxidative and ER stress (red), activation of all three MAPK subfamilies (green), translocation of NF-κB-complex into the nucleus (violet), and inflammasome inhibition (blue). Oxidative stress leads to production of mtROS and release of mtDNA and subsequent STING-dependent type I IFN (IFN-β) expression. Translocation of NF-κB-complex into the nucleus leads to expression of proinflammatory chemokines and cytokines (such as IL-8 and IL23a) and H₂O₂-dependent production of Nrf-2, which induces expression of HO-1. HO-1, heme oxygenase 1; IFN, interferon; IL, interleukin; MAPK, mitogen-activated protein kinase; mtDNA, mitochondrial DNA; mtROS, mitochondrial reactive oxygen species; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; STING, stimulator of interferon genes. Color images are available online.

Pneumococcal H₂O₂ affects inflammation in host cells

Apart from activating mtROS generation, DNA damage, and UPR in host cells, H₂O₂ secreted by Spn can also induce activation of all three mitogen-associated protein kinase subfamilies, that is, p38, JNK, and ERK, and cause nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) translocation and transcription of proinflammatory chemokines and cytokines (interleukin [IL]-8 and IL-23a) (Fig. 3) (9, 75). Canonical activation of the transcriptional regulatory factor NF-κB protein complex involves phosphorylation-induced proteolysis of the inhibitory protein IκBα, which is bound to NF-κB subunits RelA and p50 by IκB kinase. Proteolysis of IκBα leads to subsequent translocation of RelA/p50 protein complex to the nucleus (Fig. 3).

The ability of lung parenchymal cells to contribute to proinflammatory cytokine production has also been demonstrated in response to other pathogens, including Legionella pneumophila, Pseudomonas aeruginosa, Klebsiella pneumoniae, and H. influenza (22, 69, 110, 114). A mutation in pyruvate oxidase (SpxB), causing reduced H₂O₂ secretion, was shown to diminish adhesive properties of Spn to type II pneumocytes and capillary endothelial cells, which may contribute to diminished virulence in vivo (118).

Despite activating certain inflammatory pathways, pneumococcal-secreted H₂O₂ does not necessarily activate the host's immune defense. By contrast, recent results indicate that H₂O₂ released by Spn inhibits inflammasomes, key components of the innate immune system, as such further contributing to virulence and to colonization of the host (36). Thereby, H₂O₂, which is secreted in millimolar quantities by some Spn strains, has the capacity to counteract the NLRP3 inflammasome-activating effect of Ply, the main virulence factor of Spn (91). On the contrary, pneumococcus-derived H₂O₂ was shown to directly promote aggregation and activation of platelets, cells orchestrating the systemic inflammatory response (1).

Dose-dependent effects of H₂O₂ on vectorial Na⁺ transport in alveolar epithelial cells

The level of fluid in alveoli represents a critical balance between the rate of fluid movement from pulmonary capillaries and lung interstitium across the alveolar barrier into the alveolar space and the transport of salt and water out of the alveoli. Vectorial Na⁺ transport out of the alveoli by the apically expressed epithelial sodium channel (ENaC) and the basolaterally expressed Na⁺–K⁺ pump in flat type I (cover 90% of alveolar surface) and cuboidal type II alveolar epithelial cells (cover 5% of alveolar surface) is the major mechanism to maintain an optimal level of airway and alveolar surface liquid (35, 86, 87, 116, 126, 127).

ENaC, whose activity is defined by its surface expression (N) and its open probability time (Po), is critically involved in alveolar fluid clearance (AFC) (35, 87). Indeed, genetic deletion of the crucial α subunit in neonatal mice prevents them from clearing fluid from their lungs, leading to respiratory distress and death shortly after birth (66). The observation that single-nucleotide polymorphisms rs4149570 and rs7956915 of ENaC-α are associated with neonatal respiratory distress syndrome and lung fluid absorption disorders indicates that ENaC-α (SCNN1A) also has a significant role in fluid clearance in man (72). When these mechanisms become impaired, as is the case in severe pneumonia, alveolar flooding occurs, which can precipitate a lethal hypoxemia by impairing gas exchange.

Concentrations of H₂O₂ in the alveolar space in healthy individuals were estimated to be in the micromolar to tens of micromolar range (25). Physiological concentrations of endogenous, dual oxidases 1/2-mediated and exogenous H₂O₂ were shown to increase ENaC activity (39, 60, 82). This occurs at least partially through a reduction in ubiquitination of the ENaC-α subunit, which blunts the subunit's degradation and thus increases its surface expression N (33). At physiological concentrations, H₂O₂ also activates PI3-kinases that produce the anionic phospholipids, phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate, which were shown to increase the amount of time that ENaC spends in an open state, transporting Na⁺ (Po) (60, 67, 108, 147). Physiologic levels of H₂O₂ also stimulate the Na⁺–K⁺ pump in alveolar epithelial cells (29, 49, 127).

However, during pneumococcal pneumonia, near millimolar concentrations of H₂O₂ can be secreted by Spn in the alveolar space, in addition to the H₂O₂ generated by activated neutrophils and alveolar macrophages (Fig. 4). These high levels suppress ENaC-α transcription, in an ERK-dependent manner, and can inhibit the Na⁺–K⁺ pump (29, 49, 127, 131, 141). As a consequence, vectorial Na⁺ transport is impaired and alveolar flooding can occur, which can precipitate a lethal hypoxemia by impairing gas exchange (Fig. 4). Spn release of Ply can aggravate this further, since this toxin has also been shown to impair ENaC activity (77).

FIG. 4.

The complex actions of H₂O₂ in alveoli during pneumococcal pneumonia. Spn will migrate into the lower respiratory tract, and since it lacks catalase will generate millimolar levels of H₂O₂, through the actions of pyruvate oxidase (SpxB) and lactate oxidase (LctO), which will diffuse into the alveolar space (1). Moreover, an early neutrophil-mediated and a later macrophage-derived generation and secretion of μmolar levels of H₂O₂ will occur (2, 3). High ROS levels in the alveolar space will promote alveolar endothelial barrier function (4) and will impair AFC (5), which is mainly mediated through vectorial sodium transport, involving the apically expressed ENaC and the basolateral Na⁺-K⁺ pump in type II pneumocytes. AFC, alveolar fluid clearance; ENaC, epithelial sodium channel; ROS, reactive oxygen species. Color images are available online.

Impact of H₂O₂ on alveolar–capillary barrier function

A tight barrier structure of only 0.3 μm thickness separates capillary blood from alveolar gas and is a crucial interface for efficient gas exchange (133, 135). A disruption of the alveolar–capillary barrier, if not resolved, can result in alveolar flooding and poor alveolar gas exchange, both of which lead to hypoxia and hypercapnia, hallmarks of ARDS (127). During pneumococcal pneumonia, high H₂O₂ levels accumulate in the alveolar space, originating mainly from Spn secretion, and to a lesser extent also from inflammatory neutrophils and alveolar macrophages, which first generate superoxide from Nox-2, which is then further converted into H₂O₂ by SOD.

An important role of H₂O₂ in the pathogenesis of acute lung injury was first recognized by the observation that both catalase and SOD protected sheep lungs from air emboli-induced increased vascular permeability (42). Moreover, intravascular H₂O₂ challenge in intact lungs was shown to cause both capillary endothelial and alveolar epithelial barrier dysfunction, characterized by increased actin stress fiber formation, intercellular gaps, and intracellular Ca²⁺ levels. These effects on barrier function could be partially inhibited by increasing cAMP or cGMP levels (102, 115, 117).

H₂O₂ can mediate alveolar epithelial–endothelial paracrine signaling. As such, increased H₂O₂ levels in the alveolar space and pneumocytes can contribute to capillary endothelial barrier dysfunction (63). Recently, an important role for transient receptor potential (TRP) channels was suggested in the actions of ROS. This family of cation channels depolarizes the membrane potential and regulates intracellular concentrations of cations such as Ca²⁺, the latter of which is known to regulate barrier function (113). Several TRP channels, including transient receptor potential melastatin 2 (TRPM2), transient receptor potential vanilloid-4 (TRPV4), and transient receptor potential channel 6 (TRPC6), are redox sensors that can be activated by H₂O₂ (5, 32, 84, 95). Micromolar levels of H₂O₂ were shown to stimulate ADP ribose formation in human pulmonary artery endothelial cells and to activate the Ca²⁺-permeable and oxidant-activated TRPM2 channel. H₂O₂-mediated Ca²⁺ entry and diminished transendothelial electrical resistance in these cells were blunted by TRPM2 siRNA depletion or by a neutralizing antibody (59). TRPM2 is also expressed in alveolar epithelial cells, and TRPM2 knockout mice were shown to be less susceptible to bleomycin-induced lung inflammation, which is known to be accompanied by increased H₂O₂ levels (144).

Phosphorylation of TRPV4 by the Src family kinase Fyn, which is tethered to the cell membrane by the fatty acid transporter CD36, was recently shown to significantly contribute to H₂O₂-induced Ca²⁺ influx in lung microvascular endothelial cells (120). Proposed molecular mechanisms of TRPV4-mediated edema formation include Ca²⁺-induced activation of myosin light-chain kinase in endothelial cells (11) and the opening of Ca²⁺-activated K⁺ channels (KCa3.1) in epithelial cells, which induce hyperpolarization (130).

Finally, TRPC6 was shown to be critically involved in the regulation of pulmonary vascular permeability and lung edema formation during LPS or ischemia/reperfusion-induced acute lung injury (134).

Clinical significance of endogenous H₂O₂ generation during pneumococcal pneumonia

The SpxB gene found in nearly all Spn strains encodes pyruvate oxidase, an enzyme that converts pyruvate to acetyl phosphate and generates near suicidal levels of H₂O_2. These high levels of endogenous H₂O₂ would normally cause bacterial death in the absence of catalase, yet Spn is able to resist them and to even use them as a factor to outcompete S. aureus in the nose and to foster colonization in the host. As indicated above, a general strategy used by Spn and lactic-acid bacteria to avoid the toxic effects of the Fenton reaction is replacing iron with manganese. Also, the production of high levels of ferritin-binding proteins keeps free-iron levels low enough to suppress damage to DNA.

The complex role of H₂O₂ in pneumococcal-infected patients was clearly documented by recent observations with clinical isolates of serotype 1 belonging to 2 major clonal complexes (CCs), that is, CC228, associated with low mortality, and CC217, associated with high mortality (15, 121). Mutations in SpxB resulted in larger colony variants unable to produce endogenous H₂O₂. Intravenous challenge of mice with these mutants revealed that early macrophage-mediated clearance was lower than that of wild-type bacteria, and resulted in a higher bacterial load (121). Nevertheless these mutants were less efficient in the colonization process when compared with the wild-type strain. Thus, a requirement for H₂O₂ in the infection process varies as the bacteria moves from niches that are oxygen rich to those that have low oxygen tension.

Alcoholics are at increased risk for developing pneumococcal pneumonia and a role for pneumococcal H₂O₂ for this predilection has also been postulated. The Spn type 2 D39 strain, which is ethanol-tolerant due to increased alcohol dehydrogenase E (adhE) expression, exhibits increased H₂O₂ generation. Thus, ethanol-fed mice were more susceptible to infection with the D39 wild-type bacteria than with the ΔadhE strain (80).

Future Directions

Pulmonary permeability edema as a consequence of the ARDS is a main cause of morbidity and mortality of pneumococcal pneumonia. In the absence of proven pharmacologic treatments, clinicians have to rely on ventilation strategies to deliver oxygen to patients, which can sometimes make the situation worse if the ventilation pressure is too high and causes ventilator-induced lung injury. Permeability edema is characterized by hyperpermeability of the alveolar–capillary barrier, combined with a deficiency to clear liquid from the alveolar space. Vectorial Na⁺ transport through the apically expressed ENaC and the basolateral Na⁺–K⁺ pump is crucial for AFC. ARDS patients with an impaired capacity to clear alveolar fluid were shown to be at significantly higher risk to die (132).

During pneumococcal pneumonia, the pathogen can generate millimolar amounts of H₂O₂ that can rapidly diffuse through cell membranes and accumulate in the alveolar space. Whereas endogenous H₂O₂ clearly represents a colonization advantage to the pathogen, exogenous H₂O₂ in the ∼10 μM range generated by the Nox-2/MPO or Nox2/SOD pathways in neutrophils and alveolar macrophages promotes bacterial clearance. Although the detailed mechanism by which the catalase-negative pneumococci can withstand high concentrations of endogenous and phagocyte-derived exogenous H₂O₂ is incompletely understood, several well-defined enzymatic pathways in the pathogen and in phagocytic cells of the host are involved, as summarized in Figure 5.

FIG. 5.

Overview of positive (green) and negative (red) actions of endogenous and secreted H₂O₂ in the pathogen and in the host lung during pneumococcal pneumonia. In Spn, endogenous H₂O₂ promotes nucleotide, glycolytic, and capsule biosynthesis and increases Ply secretion. Secreted H₂O₂ kills competing pathogens in the respiratory tract and serves as a virulence factor, since it reduces NLRP3 inflammasome activation in the host. In the alveolar space, Spn-secreted H₂O₂ can induce activation of the UPR and cause DNA damage, which can precede apoptosis. Moreover, H₂O₂ can impair the alveolar–capillary barrier, induce inflammation, and blunt vectorial Na⁺ transport, crucial for AFC in pneumocytes. All of these actions of H₂O₂ on the host lung cells can promote the development of pulmonary edema during pneumococcal pneumonia. Ply, pneumolysin. Color images are available online.

Excessively high levels of exogenous H₂O₂ in the alveolar space during pneumococcal pneumonia can be damaging to the pathogen, they also have effects on host cells of the alveolar space. Because H₂O₂ is membrane permeant, it can promote DNA damage and apoptosis, induce the UPR and blunt inflammasome-mediated host defense, and increase inflammation. In particular, type 1 and 2 alveolar epithelial cells and the microvascular endothelial cells make up the alveolar–capillary barrier in the lungs affected by high H₂O₂ concentrations. As a consequence of reduced subunit expression, impaired Na⁺-transport by ENaC and the Na⁺–K⁺ pump in AT1/2 cells will lead to a dysfunctional AFC. Moreover, impaired barrier function in both the epithelial and endothelial compartments will promote fluid, protein, and cell accumulation in the alveolar space.

Future therapeutic approaches for pneumococcal pneumonia should potentially aim at reducing the harmful effects of ROS in lung cells, without impairing host defense. As findings with SpxB mutants have indicated, strategies to blunt endogenous H₂O₂ generation in the pathogen seem challenging. However, agents able to impair endogenous H₂O₂ generation or promote their downstream effects on iron homeostasis could decrease the harmful effects of the pathogen and improve macrophage-mediated host defense.

In view of their role in barrier function, TRP channel inhibitors could represent attractive therapeutic candidates to counteract the actions of high levels of H₂O₂ during pneumococcal pneumonia. However, their rather ubiquitous expression and the multitude of their functions (regulation of the inflammatory response, pulmonary vasomotor control, and systemic blood pressure) may render also this approach challenging.

As such, therapeutic candidates that reduce the deleterious effects of oxidative stress on barrier function and AFC specifically in the host cells, without impairing antipneumococcal host defense, might hold the key to developing a successful pharmacological approach to the potentially lethal permeability edema associated with severe pneumonia.

The alpha-tocopherol form of vitamin E was shown to boost elastase activity of human neutrophils as well as their ability to kill Spn (13). Especially noteworthy are promising preclinical data with the potent antioxidant ascorbic acid (vitamin C), which in preclinical studies was shown to attenuate systemic inflammation and blunt sepsis-induced coagulopathy and vascular injury (40, 41, 43). In a recent phase 2 clinical trial in severely ill patients with sepsis and ARDS, high-dose intravenous administration of vitamin C did not significantly improve the primary endpoint, that is, modified Sequential Organ Failure Assessment (mSOFA) score at 96 h (44). Yet, taking into account mSOFA scores from patients who died during the trial, the mSOFA score at 96 h was significantly lower in patients on the test drug than in the placebo group (45).

A tumor necrosis factor (TNF)-derived peptide—the TIP peptide (a.k.a. AP301, solnatide), which mimics the lectin-like domain of the cytokine and directly binds to the α subunit of ENaC, the latter of which is expressed in both epithelial and endothelial lung cells (27, 28), was shown to significantly reduce oxidative stress in pulmonary artery endothelial cells under hypoxia/reoxygenation and in transplanted rat lungs (55, 56). The peptide significantly blunted bacterial toxin-induced capillary leak and pulmonary edema in mice, rats, rabbits, and pigs and was moreover shown in a phase 2 clinical trial to significantly reduce extravascular lung water index in acute lung injury patients (a measure for edema) (68). Interestingly the lectin-like domain of TNF was shown not to interfere with antibacterial activities of the cytokine in a murine model of septic peritonitis (76).

In conclusion, H₂O₂ plays a complex role during pneumococcal pneumonia. On the one hand, its generation by the host's phagocytes can reduce bacterial burden. On the other hand, excessive endogenous generation of H₂O₂ by the pathogen provides a means to outcompete other pathogens inhabiting the respiratory tract and to increase resistance to exogenous levels of H₂O₂. Moreover, excessive levels of ROS can significantly impair host defense and foster the formation of permeability edema in the lungs, through the impairment of alveolar–capillary barrier function and AFC capacity.

Strategies that specifically blunt H₂O₂'s deleterious actions on the host and promote pathogen susceptibility could represent a promising approach to reduce the high mortality associated with pneumococcal pneumonia.

Abbreviations Used

Ac-P

acetyl phosphate

AdhE

aldehyde-alcohol dehydrogenase E

ADP

adenosine diphosphate

AFC

alveolar fluid clearance

AhpD

alkyl hydroperoxide reductase

ARDS

acute respiratory distress syndrome

ATF4

activating transcription factor 4

ATF6

activating transcription factor 6

ATP

adenosine triphosphate

BiP

immunglobulin heavy-chain-binding protein

Ca²⁺

calcium

cAMP

cyclic adenosine monophosphate

CAP

community-acquired pneumonia

CC

clonal complex

CcdA

cytochrome c-type biogenesis protein homologue

CD36

cluster of differentiation 36

CDC

cholesterol-dependent cytolysin

cGMP

cyclic guanosine monophosphate

CiaRH

ciaH = histidine kinase gene

ClpP

ATP-dependent Clp protease proteolytic subunit

CO₂

carbon dioxide

CodY

GTP-sensing transcriptional pleiotropic repressor

ComE

response regulator ComE

CTM

CcdA, TlpA, MsrAB

Dpr

Dps-like peroxide resistance

Dps

DNA-binding protein from starved cells

DUOX1/2

dual oxidase 1/2

eiF2α

eukaryotic translation initiation factor 2α

ENaC

epithelial sodium channel

ER

endoplasmic reticulum

ERAD

endoplasmic-reticulum-associated protein degradation

ERK

extracellular signal-regulated kinases

FabF

3-oxoacyl-[acyl-carrier-protein] synthase 2

Fyn

proto-oncogene tyrosine-protein kinase Fyn

H₂O₂

hydrogen peroxide

HO-1

heme oxygenase 1

HtrA

high-temperature requirement A

IκBα

nuclear factor of kappa light polypeptide gene enhancer in B cell inhibitor, alpha

IFN-I

type I interferon

IL

interleukin

IRE1

inositol-requiring enzyme 1

JNK

c-Jun N-terminal kinase

LctO

lactate oxidase

LPS

lipopolysaccharide

MAPK

mitogen-associated protein kinase

MerR

mercury-sensing regulatory protein

MLCK

myosin light-chain kinase

MPO

myeloperoxidase

mSOFA

modified sequential organ failure assessment

MsrAB

methionine sulfoxide reductase A/B protein

mtDNA

mitochondrial DNA

mtROS

mitochondrial ROS

Na⁺–K⁺ pump

sodium–potassium pump

ND

not determined

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

Nox-2

NADPH oxidase 2

Nrf-2

nuclear factor erythroid 2-related factor 2

O₂

oxygen

OxyR

hydrogen peroxide-inducible genes activator

PERK

protein kinase R (PKR)-like ER kinase

PerR

ferric uptake regulator

PI3-kinase

phosphoinositide 3-kinase

PIP₂

phosphatidylinositol-4,5-bisphosphate

PIP₃

phosphatidylinositol-3,4,5-trisphosphate

Ply

pneumolysin

PsaA

pneumococcal surface adhesin A

RelA

nuclear factor NF-kappa-B p65 subunit

RIDD

regulated Ire1-dependent decay

RitR

orphan response regulator

ROS

reactive oxygen species

S1P

site-1-protease

S2P

site-2-protease

siRNA

silencing RNA

SOD

superoxide dismutase

Spn

Streptococcus pneumoniae

SpxB

pyruvate oxidase

STING

stimulator of interferon genes

StkP

serine/threonine kinase

TCS04

two-component system 4

TlpA

thioredoxin-like protein family

TNF

tumor necrosis factor

TpxD

thiol peroxidase

TRP

transient receptor potential

TRPC6

transient receptor potential channel 6

TRPM2

transient receptor potential melastatin 2

TRPV2

transient receptor potential vanilloid

UPR

unfolded protein response

VILI

ventilator-induced lung injury

XBP1

X-box-binding protein 1

Author Disclosure Statement

R.L. is inventor on patents related to the use of the TIP peptide in ARDS. The other authors have no conflict of interest.

Funding Information

The study was funded by the National Institutes of Health HL134934 (Y.S.), HL125926 (D.F.), DK110409 (D.C.E.), and HL138410 (R.L.), by the Lungen- und Atmungsstiftung Bern, Switzerland (J.H., R.L.), and by the German Research Foundation SFB-TR 84 “Innate Immunity of the Lung: Mechanisms of Pathogen Attack and Host Defense in Pneumonia” (M.A.M. and T.C.).