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In vitro and limited in vivo evidence suggests that reactive oxygen species derived from NADPH oxidases (NOX-ROS) play an important role in inflammatory responses by enhancing the activity of redox-sensitive cell signaling pathways and transcription factors. Here, we investigated the role of NOX-ROS in TNFα-induced acute inflammatory responses in vivo, using mice deficient in the gp91phox (NOX2) or p47phox subunits of NADPH oxidase. Age- and body weight-matched C57BL/6J wild-type (WT) and gp91phox or p47phox knockout mice were injected i.p. with 50 μg TNFα/kg b.w. or saline vehicle control and sacrificed at various time points up to 24 hrs. Compared to WT mice, gp91phox −/− mice exhibited significantly diminished (P<0.05) TNFα-induced acute inflammatory responses in the lungs but not other tissues, including heart, liver, and kidney, as evidenced by decreased activation of the redox-sensitive transcription factor, NFκB, and decreased gene expression of interleukin (IL)-1β, IL-6, TNFα, E-selectin, and other cellular adhesion molecules. Similar results were observed in p47phox−/− mice. Interestingly, decreased lung inflammation in knockout mice was accompanied by increased leukocyte infiltration into the lungs compared to other tissues. Our data suggest that phagocytic NOX-ROS signaling plays a critical role in promoting TNFα-induced, NFκB-dependent acute inflammatory responses and tissue injury specifically in the lungs, which is effected by preferential leukocyte infiltration.
The NOX family of NADPH oxidases, a highly regulated, membrane-bound group of enzyme complexes found in a variety of phagocytic and non-phagocytic cells, is a major biological source of superoxide radicals and hydrogen peroxide (1). In phagocytic cells the core enzyme consists of five subunits: p40phox, p47phox, p67phox, p22phox, and gp91phox (NOX2). In addition, two low-molecular weight GTP-binding proteins, Rap 1A and rac1/2, are involved in activation of the enzyme. Mice that are genetically deficient in gp91phox or p47phox exhibit a reduced capacity to generate superoxide radicals (2–4).
Activation of NADPH oxidases takes place either at the plasma membrane, leading to release of superoxide radicals into the extracellular environment, or at intracellular membranes, leading to local formation of superoxide radicals and hydrogen peroxide in intracellular compartments. Phagocytic NADPH oxidase, originally described in neutrophils and macrophages, serves a critical function in host-defense against invading pathogenic microorganisms. The non-phagocytic NADPH oxidases, found in smooth musclecells, synoviocytes, chondrocytes, endothelial cells, and epithelial cells, produce smaller amounts of reactive oxygen species (ROS), which may play a critical role in cell signaling and normal cell growth and function (1). However, the mechanisms underlying intracellular ROS production and the relative concentrations and activities of NADPH oxidases in each of the non-phagocytic cell types have not been fully determined.
In recent years there has been increasing evidence indicating that ROS, and in particular hydrogen peroxide, act as mediators in cell signaling in a broad array of physiological and pathophysiological responses (1, 5, 6). The regulation of gene transcription of pro-inflammatory cytokines, chemokines, and cellular adhesion molecules has been shown to be mediated by reduction-oxidation (redox) sensitive signaling pathways and transcription factors, such as nuclear factor κB (NFκB) and activator protein-1 (AP-1) (7, 8). NFκB activation leads to upregulation of a wide array of inflammatory genes critical for neutrophilic inflammation and host defense against infectious agents, and may also cause the systemic inflammatory response syndrome (9, 10). A variety of in vitro studies has suggested that ROS act as second messengers affecting NFκB activation (11–13). Thus, it is possible that NADPH oxidase-derived ROS (NOX-ROS) play an important role in regulating cell signaling and host inflammatory responses in both phagocytic and non-phagocytic cells. However, there is limited and controversial evidence from in vivo studies to support this notion (3, 4, 14–21).
Tumor necrosis factor α (TNFα) is a major mediator of inflammation, immunity, and apoptosis, and has been implicated in the pathogenesis of a wide spectrum of human diseases (22, 23). It promotes inflammation by increasing blood neutrophil concentrations, enhancing expression of multiple inflammatory mediators, and inducing adhesion of neutrophils and monocytes to vascular endothelium and their subsequent tissue infiltration. Through binding to its cellular receptors, TNFα activates multiple signaling pathways (23, 24); ROS have been implicated in mediating TNFα signaling, although their source has not been fully established (5). Recent evidence indicates that NOX-ROS contribute to TNFα-induced NFκB activation and adhesion molecule and IL-8 expression in cultured endothelial cells (14, 25–28). However, the role of NOX-ROS in cell signaling and gene expression in TNFα-induced acute inflammatory responses in vivo remain to be fully determined.
We have recently shown that genetic deficiency of gp91phox or p47phox does not diminish, but rather enhances, LPS-induced acute inflammatory responses in vivo, suggesting that NOX-ROS do not play a role in enhancing inflammation, but rather in its resolution (21). As the signaling pathways in TNFα-induced acute inflammation are distinct from those triggered by LPS, in the current study we investigated whether limiting NOX-ROS production by genetic deficiency of gp91phox or p47phox is associated with impairment or enhancement of TNFα-induced acute inflammatory responses in vivo.
Age- and body weight-matched female C57BL/6J WT, gp91phox−/−, and p47phox−/− mice at 10–12 weeks of age and weighing 20–22 g were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were housed under specific pathogen-free conditions and in a temperature and humidity-controlled environment (12-hr light/dark cycle) with unlimited access to tap water and food. Animals were acclimated for 7–10 days before initiation of experiments. All animal procedures were reviewed and approved by the Oregon State University Institutional Animal Care and Use Committee.
Wild-type, gp91phox−/−, and p47phox−/− mice were randomly assigned to receive i.p. injection of the vehicle HBSS (Sigma-Aldrich, St. Louis, MO) as controls, or 50 μg of murine recombinant TNFα (R&D Systems, Minneapolis, MN) per kg body weight (b.w.), and sacrificed 1, 3, 8, or 24 hrs after injections. Blood and various tissues were collected for further analysis.
Serum concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1), and TNFα were measured by quantitative colorimetric ELISA kits from R&D Systems. The reported sensitivities of the assays are 5 pg/ml sVCAM-1, 29 pg/ml sICAM-1, 2 pg/ml MCP-1, and 5 pg/ml TNFα.
Total RNA was isolated from different tissues using TRIzol Reagent (Invitrogen, Carlsbad, CA). cDNA synthesis was performed using the high capacity cDNA archive kit from Applied Biosystems (Foster City, CA). mRNA levels of E-selectin, VCAM-1, ICAM-1, MCP-1, TNFα, IL-1β, IL-6, myeloperoxidase (MPO), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantitated by real-time PCR. All primers and probes were purchased as kits from Applied Biosystems. The assays are supplied as a 20x mixture of PCR primers and TaqMan minor groove binder 6-FAM dye labeled probes with a nonfluorescent quencher at the 3′ end. TaqMan quantitative PCR (40 cycles of 95°C for 15 seconds and 60°C for 1 min) was performed using TaqMan Universal PCR Master Mix (Applied Biosystems) in 96-well plates with the ABI Prism 7500 Sequence Detection System (Applied Biosystems). To obtain relative quantitation, two standard curves were constructed in each plate with one target gene and an internal control gene (GAPDH). Standard curves were generated by plotting the threshold cycle number values against the log of the amount of input cDNA and used to quantitate the expression of the various target genes and GAPDH gene in the same sample.
Mice were sacrificed eight hours after HBSS (control) or TNFα injection. The left lung was tied off, and the right lung was lavaged by intratracheal injection of two sequential 0.6-ml aliquots of HBSS, followed by aspiration. Recovered fluid was pooled and centrifuged. Supernatants were preserved for measurement of total albumin concentration using an enzyme-linked immunoassay (Bethyl Labs, Montgomery, TX). The leukocyte pellet was resuspended in HBSS and cell numbers were counted using a hemocytometer.
Mice were sacrificed eight hours after HBSS (control) or TNFα injections. The left lung was removed and fixed with 10% neutral-buffered formalin and embedded in paraffin. After dewaxing and rehydration, 5-μm-thick paraffin sections were stained with hematoxylin and eosin. Neutrophils were counted from10 randomly selected fields (20x magnification, 0.4×0.4 mm2) in each section. A total of 30 fields were screened from sections selected from the upper, middle, and lower parts of the left lung.
Lung tissue samples were homogenized in 1 ml lysis buffer (Cell Signaling Technology, Danvers, MA) followed by centrifugation at 14,000 g and 4°C for 15 min. The supernatant containing solubilized proteins was recovered, frozen in aliquots, and stored at −80°C until analysis. Protein samples were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated overnight with mouse anti-MPO antibody (R&D systems) or rabbit anti-α-tubulin antibody (Cell Signaling), after blocking with 0.1% Tween 20 in Tris-buffered saline containing 5% skim milk powder. The membranes were incubated with HRP-conjugated goat anti-mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-rabbit antibody (Cell Signaling) for 1 hr at room temperature. The proteins were detected by enhanced chemiluminescence (ECL™, GE Healthcare, Buckinghamshire, UK).
Lung tissue samples were homogenized in 50 mM phosphate buffer (pH 7.4) and centrifuged at 10,000 g for 10 min at 4°C, and the resulting pellet was resuspended in 50 mM potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich). The suspension was subjected to three cycles of freezing and thawing and disrupted further by sonication (40 s). Subsequently, the samples were centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was used for measuring MPO activity with the Myeloperoxidase Activity Assay Kit from Northwest Life Science Specialties (Vancouver, WA). One unit of MPO activity is defined as the amount of enzyme that catalyzes the formation of 1 nM taurine chloramine from taurine at pH 6.5, 25°C, over 30 min in the presence of 100 mM chloride and 100 μM H2O2. The MPO activity was normalized to the total protein concentration.
Nuclear extracts were prepared from different tissues using nuclear extract kits (Active Motif, Carlsbad, CA). For analysis of activation of nuclear transcription factors, ELISA-based assays (Active Motif) were used to determine the DNA binding activity of NFκB (p65) and AP-1 (c-fos). The specificity of binding was confirmed by competition with either wild-type or mutant oligonucleotides. The wild-type oligonucleotides prevent NFκB and AP-1 binding to the probe immobilized on the plates. Conversely, the mutated oligonucleotides should have no effect on NFκB and AP-1 binding activity.
The data were calculated as means ± SEM and analyzed by unpaired student’s t-test or ANOVA followed by Holm-Sidak post-hoc test. Statistical significance was set at P<0.05.
To assess the time-course of TNFα-induced acute, systemic inflammation in mice, serum levels of selected inflammatory mediators were measured up to 24 hrs after i.p. injection of TNFα (50 μg/kg b.w.). As shown in Fig. 1, TNFα treatment caused a rapid increase in serum concentrations of sVCAM-1, sICAM-1, MCP-1, and TNFα: TNFα peaked at 1 hr, while the other inflammatory mediators were significantly increased at 3 hrs. sVCAM-1 and sICAM-1 remained elevated up to 24 hrs, while MCP-1 and TNFα returned to baseline at 8 hrs. Interestingly, these TNFα-induced changes in serum levels of inflammatory mediators were indistinguishable between gp91phox−/− and WT mice at all time points examined (Fig. 1). Similar results were observed in serum of p47phox−/− mice 3 hrs after TNFα injection (data not shown). These data suggest that NADPH oxidase activity is not required for normal TNFα-induced systemic inflammatory responses.
To assess TNFα-induced gene expression of inflammatory mediators, their mRNA levels were measured in lung, heart, liver, kidney, small intestine, and spleen. As expected, lung mRNA levels of all inflammatory mediators increased in a time-dependent manner following TNFα administration to WT mice (Fig. 2). VCAM-1, ICAM-1, and IL-6 mRNA levels were increased substantially 1 hr after TNFα injection and remained elevated for at least 2 hrs before declining to baseline after 8 hrs. E-selectin, TNFα, and IL-1β mRNA levels peaked at 1 hr and then rapidly declined at 3 hrs, returning to baseline after 8 hrs (Fig. 2).
Interestingly, TNFα-induced upregulation of inflammatory gene expression in lung was significantly diminished in gp91phox−/− mice compared to WT mice (Fig. 2). Hence, mRNA levels of E-selectin at 1, 3, and 24 hrs, IL-1β at 1 hr, and VCAM-1, ICAM-1, TNFα, and IL-6 at 3 hrs were significantly lower in gp91phox−/− mice compared to WT mice (P<0.05, n=4–5). These data indicate that genetic deficiency of gp91phox decreases TNFα-induced acute inflammatory responses in the lungs, consistent with the notion that NOX-ROS play an important role in this process.
Exposing WT animals to TNFα for 3 hrs strongly up-regulated expression of all inflammatory genes measured in all tissues examined (Table 1). Compared to WT mice, inflammatory gene expression was significantly lower in the lungs of gp91phox−/− and p47phox−/−mice (Figs. 2 and and3)3) but, interestingly, not any other tissues (Table 1). Hence, in gp91phox−/− mice, lung mRNA levels of E-selectin, VCAM-1, ICAM-1, IL-1β, IL-6, and TNFα were diminished by 72%, 63%, 61%, 35%, 57%, and 51%, respectively (P<0.05, n=4–5) (Fig. 2). Similar results were observed in lung tissue of p47phox−/− mice (Fig. 3). However, lack of gp91phox or p47phox did not diminish TNFα-induced upregulation of these inflammatory genes in any other tissues (Table 1). To the contrary, TNFα-induced IL-6 and TNFα gene expression seemed to be further enhanced in liver of gp91phox−/−mice (Table 1A).
It is well documented that the redox-sensitive transcription factors, NFκB and AP-1, play critical roles in TNFα-induced upregulation of many inflammatory genes. We observed that TNFα treatment increased NFκB-DNA binding activity in a time-dependent manner in the lungs of WT mice. NFκB binding activity increased substantially 1 hr after TNFα injection, peaked at 3 hrs, and returned to baseline after 8 hrs (Fig. 4A). Interestingly, TNFα-induced NFκB activation was significantly lower at both the 1 and 3-hr time points (by 43% and 57%, respectively) in the lungs of gp91phox−/− mice compared to WT mice (P<0.05, n=4–5) (Fig. 4A and B). Treatment of WT mice with TNFα for 3 hrs also strongly induced NFκB activity in various other tissues (Fig. 4B and C). However, TNFα-induced NFκB activation was not significantly lower in non-lung tissues of gp91phox−/− mice (Fig. 4B), as well as p47phox−/− mice (Fig. 4C), compared to WT mice (P<0.05, n=4–5). Lack of gp91phox and p47phox did not, however, diminish TNFα-induced activation of AP-1 in all tissues examined compared to WT mice (data not shown).
Bronchoalveolar lavage (BAL) fluid was used for assay of total albumin concentration and total neutrophil count as independent assessments of acute lung injury and alveolar inflammation. The TNFα-induced increase in albumin concentration in BAL fluid was significantly less in gp91phox−/− mice compared to WT mice (231±23 μg/ml v.s. 306±20 μg/ml, P<0.05, n=5). In contrast, a significant increase in lavage neutrophil count was observed in TNFα-treated gp91phox−/− mice compared to WT mice (21.2±3.3×104 cells vs. 7.4±0.9×104 cells, P<0.05, n=5), while there was no significant difference between HBSS-treated (control) gp91phox−/− mice and WT mice (4.6±1.0×104 cells vs. 4.4±0.3×104 cells, n=5).
Since polymorphonuclear neutrophils (PMNs) and other phagocytic cells, such as monocyte-macrophages, play critical roles in acute inflammation and tissue injury, the specific enzyme marker, myeloperoxidase, was measured in various tissues after TNFα challenge. As shown in Fig. 5A, TNFα treatment induced time-dependent upregulation of MPO gene expression in the lungs of both gp91phox−/− and WT mice, which peaked at 3 hrs and remained elevated for up to 8 hrs, and then declined after 24 hrs. MPO mRNA levels also increased in other tissues, such as heart, liver, and kidney, albeit to a much lesser degree than in lung (Fig. 5B and C). Interestingly, TNFα-induced changes in MPO gene expression were indistinguishable between gp91phox−/−, p47phox−/−, and WT mice (Fig. 5A, B, and C).
Consistent with increased MPO message levels, MPO protein levels (Fig. 5D) and activity (Fig. 5E) in lung also were significantly increased 8 hrs after TNFα treatment, and indistinguishable between gp91phox−/− and WT mice. Furthermore, morphological analysis showed significantly increased numbers of neutrophils in the lungs of gp91phox−/− mice compared to WT mice (P<0.05) (Fig. 6A, B, and E). TNFα treatment further markedly induced accumulation of abundant interstitial neutrophils in both gp91phox−/− and WT mice; however, there was no significant difference between these two groups of animals (Fig. 6C, D, and E). These data indicate that the lungs are the main target for activated PMNs, and genetic deficiency of phagocytic NADPH oxidase does not impair PMN chemotaxis during TNFα-induced acute inflammation.
Increased ROS production and oxidative stress contribute to cellular and tissue injury during the development of inflammatory diseases (1, 5, 10, 29, 30). Evidence also suggests that ROS modulate a wide variety of cell-signaling processes (1, 5, 6). Yet, the relationship between oxidative stress and the pathology of inflammatory diseases is not fully understood, largely because of limited understanding of the mechanisms by which ROS function as signals in physiological or pathophysiological settings. Recent studies, though still controversial, have suggested that NOX-ROS contribute to oxidative stress and may serve as cell signaling molecules enhancing acute inflammatory responses and tissue injury (1, 3, 4, 14–21). However, the role of NOX-ROS in TNFα-induced cell signaling and acute inflammatory responses in vivo is less clear.
In the present study, we found that genetic deficiency of gp91phox or p47phox significantly attenuated TNFα-induced NFκB activation, upregulation of inflammatory gene expression, and acute tissue injury in the lungs but not other tissues of mice. These effects were associated with higher levels of leukocyte infiltration into the lungs compared to other tissues. Therefore, our data provide new evidence that both NOX-ROS dependent and independent pathways are involved in TNFα signaling, and especially phagocytic NOX-ROS play an important role in TNFα-induced acute inflammatory responses in the lungs.
It is now widely accepted that increased ROS production during acute lung inflammation largely originates from phagocytic NOX2-expressing cells within the respiratory tract, including alveolar macrophages and other resident or infiltrating inflammatory-immune cells, including neutrophils, eosinophils, and lymphocytes. However, direct evidence that NOX2 activation contributes to respiratory disease pathology is sparse. Although NOX2 in phagocytic cells primarily functions in host defense by generating antimicrobial ROS, activation of NOX2 in neutrophils was also found to regulate intracellular signaling pathways that modulate neutrophil (29) and endothelial (14) inflammatory gene expression via NFκB activation. Similarly, stimulation of macrophage NOX2, which produces markedly less ROS compared to neutrophils, has been linked to regulation of NFκB and AP-1 signaling and production of pro-inflammatory cytokines (30). In fact, accumulation of infiltrating neutrophils is a key event in the onset and development of inflammation and resultant host tissue injury in the lungs.
Our data showed that treatment of animals with TNFα strongly increased gene expression, protein levels, and enzymatic activity of MPO, a biomarker of neutrophils, in the lungs but not other tissues, such as heart, liver, and kidney. Furthermore, TNFα-induced increases of lung MPO and accumulation of interstitial neutrophils in the lungs were indistinguishable between NADPH oxidase knockout mice and WT mice. However, deficiency of NOX2 or p47phox significantly limited TNFα-induced activation of NFκB and expression of inflammatory genes. These data indicate that lung is the main target tissue for activated neutrophils during the acute phase of inflammation, and phagocytic NADPH oxidase critically contributes to TNFα-induced inflammatory responses in the lungs. These findings are consistent with previous studies showing that neutrophil NADPH oxidase plays a significant role in the mechanism of TNFα-induced NFκB activation and ICAM-1 expression in the lungs of p47phox−/− and WT mice (14).
Acute lung injury is characterized clinically by derangements in lung vascular permeability, which is a cardinal feature of inflammation and the initial cause of the profound physiological abnormalities observed in patients with acute lung injury (31). In the present study we found in our murine models that deficiency of NOX2 significantly attenuated the TNFα-induced increase in total albumin concentration in BAL fluid, which reflects leakage of protein into the alveolar space across both the vascular and epithelial barriers. The cumulative effects of NOX2 knock-out on TNFα-induced lung injury could account for both a decrease in vascular leakage and a reduction in inflammation characterized by reduced lung NFκB activation and inflammatory gene expression, despite the fact that lung interstitial neutrophil counts were similar between TNFα-treated NOX2 knock-out and WT mice. These data indicate that neutrophils lacking a functional NADPH oxidase have normal chemotactic activity but a reduced ability to mount an inflammatory response to TNFα in the lungs, suggesting that phagocytic NADPH oxidase critically contributes to TNFα-induced acute lung injury.
Although it is known that ROS affect a number of signaling pathways by activating or inactivating specific protein kinases and phosphatases, respectively (1), the signaling molecules specifically targeted by ROS in the NFκB pathway have not been unequivocally identified. While some studies have found that ROS act as second messengers and are involved in TNFα signaling and NFκB activation (7–12, 14), others have not found such evidence (32, 34) or even inhibition of NFκB activation by hydrogen peroxide (33). It is likely that ROS-mediated NFκB activation is restricted to certain cell types and incubation conditions, such as the types and concentrations of ROS or pro-inflammatory cytokines used. In addition, most of the evidence for a role of ROS in NFκB activation is derived from in vitro study, and in vivo evidence is scant. Our in vivo data suggest that TNFα-induced NFκB activation in lung is indeed enhanced by NOX-ROS (Scheme 1), as it was significantly diminished in gp91phox−/− and p47phox−/−mice compared to WT mice. In contrast, TNFα-induced activation of AP-1, another critical transcription factor for inflammatory gene expression, was not affected by NADPH oxidase deficiency.
Our data further suggest that in non-lung tissues, NOX-ROS do not play a significant role in TNFα-mediated NFκB activation and inflammatory gene expression. In this NOX-ROS independent pathway, TNFα binding to the TNF receptor 1 (TNF-R1) triggers a series of intracellular signals causing activation of IκB kinase (IKK), which results in phosphorylation and subsequent ubiquitylation and degradation of IκB and translocation of NFκB to the nucleus (Scheme 1). In vitro evidence suggests that TNF-R1 stimulation also induces NADPH oxidase activity via PKC-mediated phosphorylation of p47phox and activation of Rac1, thereby increasing ROS production and enhancing IKK and NFκB activation (35, 36). However, our data suggest that this mechanism does not play a significant role in vivo, unless extensive neutrophil infiltration occurs and ROS are generated in large quantities by NOX2, such as in the lungs of TNFα-exposed mice. It is possible that these PMN-derived ROS, in particular hydrogen peroxide which is relatively long-lived and membrane-permeable are transmitted into resident tissue cells and serve as signaling molecules activating IKK and NFκB (Scheme 1). These exogenous ROS generated by NOX2 of infiltrating PMNs seem to be more critical in mediating TNFα-induced inflammatory responses in vivo than ROS signals generated endogenously by NOX in resident macrophages or non-phagocytic cells (14). Nevertheless, we cannot exclude the possibility that ROS generated from other cellular sources, such as xanthine oxidase, mitochondria, or cytochrome P450, may have played a role in TNFα-induced cell signaling and inflammation in non-lung tissues (5, 37).
It should be noted that the results from previous studies for a role of NOX-ROS in lung inflammation in vivo are still controversial. It has been shown that LPS or Pseudomonas aeruginosa-induced NFκB activation in lung tissue was impaired in p47phox−/− and gp91phox−/− mice (3, 4, 16), and LPS-induced lung inflammation in Nrf2−/−mice was significantly reduced by genetic ablation of gp91phox (38), supporting an important role of NOX-ROS in promoting inflammation. However, other investigators found no differences between p47phox−/− and WT mice in LPS-induced lung injury, alveolar damage, and infiltration of neutrophils and monocytes (17), and no amelioration of pulmonary damage in Pneumocystis-infected gp91phox−/− mice compared to WT mice (18). In fact, more recent studies found that genetic deficiency of NADPH oxidase was in many cases associated with enhanced lung inflammation and injury (19–21, 39–44), indicating that NADPH oxidase has beneficial functions in limiting inflammatory-immune responses or enhancing their resolution. For example, infection of gp91phox−/− mice with the yeast C. neoformans or the influenza virus was found to result in increased Th1-skewed inflammation and granuloma formation (42, 43). Deficiency of gp91phox was also reported to markedly increase pulmonary cytokine and chemokine expression and neutrophil recruitment in a mouse model of pneumococcal pneumonia (20), and lung levels of pro-inflammatory mediators, such as keratinocyte chemoattractant, IL-6, MCP-1, and TNFα, were significantly increased in both gp91phox and p47phox -deficient mice in response to cigarette smoke exposure (19). Consistent with these data, we have recently shown that genetic deficiency of NADPH oxidase does not diminish, but rather enhances, LPS-induced acute lung inflammatory responses (21). These results might also be related to clinical observations in patients with the X-linked form of chronic granulomatous disease (gp91phox deficiency), who are more prone to infection and present with hyper-inflammatory conditions (45–47).
However, in the current study we showed that reduced phagocytic NOX-ROS diminished TNFα-induced acute lung inflammatory responses and lung injury, which is opposite to our observations in mice exposed to LPS instead of TNFα (21). These apparently discrepant results highlight the above notion that the role of ROS in cell signaling and inflammatory gene expression can vary widely depending on, e.g., the pro-inflammatory cytokine or bacterial agent used and the affected tissue or cell type. More studies are needed to increase our understanding of NOX-ROS-mediated cell signaling, in particular the cellular location of NOX and its association with target cells or proteins. In conclusion, our data suggest that phagocytic NOX-ROS signaling plays a critical role in promoting TNFα-induced, NFκB-dependent acute inflammatory responses and tissue injury in the lungs, whereas NOX-ROS independent pathways play a dominant role in non-lung tissues.
This publication was made possible by Grant Number P01 AT002034 (BF/WJZ) from the National Center for Complementary and Alternative Medicine (NCCAM). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCCAM or the National Institutes of Health.