Author response: Female resistance to pneumonia identifies lung macrophage nitric oxide synthase-3 as a therapeutic target

Field: Medicine (Health Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract To identify new approaches to enhance innate immunity to bacterial pneumonia, we investigated the natural experiment of gender differences in resistance to infections. Female and estrogen-treated male mice show greater resistance to pneumococcal pneumonia, seen as greater bacterial clearance, diminished lung inflammation, and better survival. In vitro, lung macrophages from female mice and humans show better killing of ingested bacteria. Inhibitors and genetically altered mice identify a critical role for estrogen-mediated activation of lung macrophage nitric oxide synthase-3 (NOS3). Epidemiologic data show decreased hospitalization for pneumonia in women receiving estrogen or statins (known to activate NOS3). Pharmacologic targeting of NOS3 with statins or another small-molecule compound (AVE3085) enhanced macrophage bacterial killing, improved bacterial clearance, and increased host survival in both primary and secondary (post-influenza) pneumonia. The data identify a novel mechanism for host defense via NOS3 and suggest a potential therapeutic strategy to reduce secondary bacterial pneumonia after influenza. https://doi.org/10.7554/eLife.03711.001 eLife digest Pneumonia is a disease that is commonly caused by a bacterial infection and results in the lungs becoming inflamed. Pneumonia is a serious condition and can lead to hospitalization and sometimes death. However, women—and other female animals—are less likely than males to get pneumonia and are more likely to survive if they do. Understanding this sex-based difference may help to develop treatments or preventive actions that either reduce the number of people who get pneumonia or help infected patients to recover. Bacteria from the nose—including those that cause pneumonia—frequently enter the lungs during sleep. Luckily, the body has very robust defense mechanisms against such invasions; the immune system immediately deploys cells called macrophages as a 'first response' to devour and kill invading bacteria in the lungs. However, this system is not perfect, particularly if an individual has a weakened immune system or if they are already suffering with a respiratory infection. Indeed, many individuals with severe influenza infections are hospitalized as a result of pneumonia. Yang et al. studied why females are more able to fend off pneumonia and found that estrogen, the main female sex hormone, boosts the ability of the macrophages to kill bacteria. Treating male mice with estrogen also boosted their immune system's ability to kill off bacteria in the lungs. Investigating further, Yang et al. found that the estrogen worked by increasing the number of proteins produced from one gene called NOS3. Female mice lacking NOS3 proteins lost their pneumonia-fighting advantage. A widely used class of drugs called statins, which are used to treat cardiovascular disease, boosts the activity of the NOS3 gene. Yang et al. therefore wondered whether treatment with either estrogen or statins might prevent pneumonia, or help patients with pneumonia fight off the infection. Using a large database of information about healthcare in Denmark, Yang et al. assessed the relationship between taking these drugs and the risk of pneumonia. When several confounding factors (such as unrelated diseases that the patient was suffering from) are taken into account, the data show that the women were less likely to be hospitalized for pneumonia if they were taking statins or estrogens. Those taking both treatments had an even lower risk. Yang et al. also found that treating mice with statins or an experimental drug that boosts NOS3 activity increased the ability of the animals to fight off pneumonia-causing bacteria—even if they also had influenza—and increased the likelihood that mice already infected with pneumonia would survive. Further studies will be needed to determine if statins or the experimental drug might also help to prevent pneumonia in human patients with influenza. https://doi.org/10.7554/eLife.03711.002 Introduction Bacterial pneumonia remains a major cause of morbidity and mortality (Mizgerd, 2006; Shrestha et al., 2013). One approach to the problem might be to enhance innate immunity to infection. Normal host defenses are already quite robust, albeit imperfect, as they keep the incidence of pneumonia much lower than possible given the normal nocturnal aspiration of nasopharyngeal bacteria, example Streptococcus pneumoniae (Gleeson et al., 1997; Dockrell et al., 2012; Donkor, 2013). The resident alveolar macrophage (AM) functions as a 'first responder' phagocyte, ingesting and killing inhaled bacteria (Green and Kass, 1964; Fels and Cohn, 1985; Hussell and Bell, 2014). The importance of this function of AMs is indicated by greater susceptibility to infection and diminished bacterial clearance after their experimental depletion (Dockrell et al., 2003; Ghoneim and McCullers, 2013) or their impairment by clinical risk factors such as recent influenza infection (Sun and Metzger, 2008). To guide investigation of possible targets to improve or restore lung macrophage antibacterial function, we sought to exploit the natural experiment of gender differences in resistance to infections. Experimental models find that female mice show greater systemic resistance to pneumococci (Weiss et al., 1973; Kadioglu et al., 2011) and to many other (but not all) pathogens (McClelland and Smith, 2011). Epidemiologic studies of human pneumonia observe a greater incidence of community-acquired pneumonia in males (Gutiérrez et al., 2006) and show that males are at greater risk than females for pneumonia after admission to hospital after adjusting for other risk factors such as smoking and alcohol use (Offner et al., 1999; Andermahr et al., 2002; Gannon et al., 2004). We chose to address this problem by using a model of pneumococcal infection that approximates the frequent challenge to lung defenses by small numbers of bacteria and comparing responses in male and female mice. We identified greater female resistance to infection, mediated in large part by estrogen-dependent activation of constitutive AM nitric oxide synthase-3 (NOS3). Pharmacologic agents that enhance NOS3 function improved resistance in mouse models of both primary lung infection and post-influenza secondary pneumonia, suggesting a strategy to enhance resistance to common and serious lung infections. Results Superior resistance of female mice to pneumococcal pneumonia We tested effects of a relatively small bacteria inoculum size to simulate the common, low-level challenge to the lungs from aspiration of upper airway bacteria. Female mice and estrogen-treated male mice showed greater clearance of bacteria from the lungs and less acute inflammation (neutrophil influx) compared to normal or sham-treated males 24 hr after inoculation of S. pneumoniae (Figure 1A,B). Pilot experiments compared efficiency of delivery in male vs female mice by measuring bacterial CFUs 5 min after instillation. The results showed slightly greater initial bacterial loads in female mice (p < 0.02, n = 12/group), indicating that the female advantage does not reflect a lower inoculum due to anatomic or size differences. Greater female resistance was also observed in longer duration survival studies (Figure 1C). The gender differences we observed with 105 colony-forming units (CFU) were not seen if a lethal inoculum (∼11-fold higher) of bacteria was used, as both genders showed markedly increased inflammation and lung cytokine levels (Figure 1D–G). Figure 1 with 1 supplement see all Download asset Open asset Females show greater resistance to pneumococcal pneumonia. (A) Twenty-four hours after intranasal (i.n.) inoculation of S. pneumoniae (∼105 CFU), lung samples from female mice (and estrogen-treated male mice via subcutaneous slow-release 17-beta-estradiol pellets, ∼70 µg/day) contain fewer live bacteria than seen in male mice (n > 12, * = p < 0.01 vs control or sham-treated males) and (B) show less acute inflammation (BAL neutrophils, n > 12, * = p < 0.01). (C) After i.n. pneumococcus, female mice show significantly greater survival than male mice (2.5 × 105 CFU, n > 24, * = p < 0.01). Gender differences in pneumonic inflammation are seen with low (4 × 105 CFU), but not high (11 × 105), bacterial inocula, measured as BAL neutrophilia (D) or BAL cytokines TNF (E), MIP-2 (F), or IL-6 (G), (n > 3, * = p < 0.05). https://doi.org/10.7554/eLife.03711.003 Superior bacterial killing by female alveolar macrophages To compare the innate antibacterial function of alveolar macrophages from both genders, we measured phagocytosis and killing of pneumococci in vitro by AMs from normal mice and humans. Analysis of bacterial binding and internalization showed no differences between male and female murine AMs (Figure 2A,B; similar data with human AMs not shown). In contrast, killing of internalized bacteria was greater in female AMs than male AMs in mouse samples challenged with S. pneumoniae, as well as with other lung pathogens Staphylococcus aureus, and Escherichia Coli (Figure 2C–E). Similarly, normal human female AMs showed greater killing of ingested pneumococci than their male counterparts (Figure 2F). Figure 2 Download asset Open asset Female alveolar macrophages show better killing of ingested bacteria. Binding (A) and internalization (B) of S. pneumoniae in normal male and female AMs is similar. Female AMs kill more internalized bacteria than male AMs in assays using pneumococci (C) (n > 11, * = p < 0.01), S. aureus (D) or E. coli (E), (n > 3, * = p < 0.01). (F). Normal human female AMs also show greater killing of internalized pneumococci, (n > 5, * = p < 0.01). https://doi.org/10.7554/eLife.03711.005 NOS3 mediates the female advantage Mechanisms for killing phagocytosed bacteria include production of reactive oxygen species (ROS), example, superoxide produced by NADPH oxidase, and reactive nitrogen intermediates (RNI), example nitric oxide produced by nitric oxide synthases (NOS). We first used mice genetically deficient in NADPH oxidase (Morgenstern et al., 1997) to more directly evaluate the importance of this pathway in vivo. Bacterial clearance in NADPH oxidase-null mice was significantly impaired, but did not eliminate the gender difference, since both male and female mice were affected approximately equally (Figure 3A). Additional experiments showed no difference in generation of ROS by male and female AMs stimulated to undergo a respiratory burst in vitro (Figure 1—figure supplement 1). Similarly, quantitative real-time PCR using primers for NADPH oxidase components (phox22, 47, 91) or myeloperoxidase showed no difference in expression (data not shown). Figure 3 Download asset Open asset NOS3 and female resistance to pneumococcal pneumonia. (A) NADPH oxidase deficient (phox91−/−) mice show comparable reduction in bacterial clearance in both male and female mice (n = 6, * = p < 0.01 vs wild-type). (B) In vitro killing of pneumococci by normal mouse female AMs is inhibited by the non-selective NOS inhibitor nitro-L-arginine (NLA), but not by its inactive stereo-isomer, nitro-D-arginine (NDA), nor by the type 2 NOS specific inhibitor 1400W (n = 3–4, * = p < 0.01). (C) Female AMs from Nos3−/− mice lose the in vitro killing advantage of wild-type female AMs and show the same killing rate as wild-type or NOS3 deficient male AMs (n = 3, * = p < 0.01 vs wild-type). (D) In vivo, absence of NOS3 reduces, but does not completely eliminate, the female advantage in bacterial clearance (n = 15, * = p<0.015 vs all 3 other groups) and results in increased mortality from pneumococcal pneumonia (E) (n = 12 female mice per group, * = p < 0.01). Conversely, transgenic male mice with increased expression of human NOS3 show enhanced killing of S. pneumoniae in vivo (F) (lower bacterial survival, n > 5, * = p < 0.01). In this low-dose inoculum model, NOS2 deletion (G) or inhibition (H) causes reduced bacterial clearance in male, but not female mice (n = 8, * = p < 0.05). https://doi.org/10.7554/eLife.03711.006 To explore the potential role of RNI, we tested the effect of NOS inhibitors on bacterial killing by female murine AMs in vitro. As shown in Figure 3B, the non-selective NOS blocker nitro-l-arginine (NLA) caused substantial inhibition of female AM killing, while its stereoisomer controls NDA and the inducible NOS (iNOS or NOS2)-specific inhibitor 1400W had no effect. This suggested a role for another NOS isoform, namely the constitutively expressed endothelial type NOS3 found in alveolar and other macrophages (Miles et al., 1998; van Straaten et al., 1998; Connelly et al., 2003). This postulate was directly tested using mice genetically deficient in NOS3. In vitro, deficiency in NOS3 did not alter the killing by male macrophages, but completely abolished the female advantage in bacterial killing (Figure 3C). In vivo, absence of NOS3 had minimal effects on male clearance of bacteria, but reduced the female advantage by ∼50% (Figure 3D). Genetic deletion of NOS3 greatly reduced survival in female mice with pneumonia (Figure 3E), and transgenic overexpression of NOS3 increased bacterial clearance in male mice (Figure 3F), supporting the functional importance of this pathway. The 24 hr in vivo bacterial clearance assay provides enough time for induction of NOS2, which is known to mediate macrophage antimicrobial function in rodents (Nathan and Shiloh, 2000). To test the contribution of this isoform, we measured bacterial clearance in NOS2-deficient mice and in mice treated with the NOS2 inhibitor 1400W. The data show that male mice do rely on NOS2, since clearance of pneumococci was markedly worse in NOS2-deficient or 1400W-treated mice compared to controls (Figure 3G,H). In contrast, female mice showed a trend for improved clearance when Nos2 was genetically deleted (Figure 3G); similar results were seen in wild-type female mice after pharmacologic inhibition of NOS2 (Figure 3H). Since NO itself can inhibit NOS3 function (Buga et al., 1993; Erwin et al., 2005), the data suggest that low-level basal NOS2 or NOS2 induction actually hampers the beneficial activity of NOS3 in females. Estrogen effects on NOS3 To facilitate biochemical analysis of estrogen effects on macrophage function, we tested the effect of estrogen on bacterial uptake and killing by macrophage cell lines. The cell lines were cultured using hormone-free serum. After estrogen treatment, the macrophage cell lines J744A.1 (murine) and U937 (human) showed increased killing of internalized bacteria (Figure 4A,B), although no differences in binding or internalization of bacteria were observed (data not shown). This increased killing capacity was inhibited by the NOS inhibitors NLA or L-NMMA, but not their stereoisomer controls. After estrogen treatment (0.2 ng/ml, 1 hr), we measured slight, albeit consistent, increases in nitrite production by bacteria-challenged J774A.1 macrophages (p < 0.01, paired t-test, n = 13). The increase (∼25 pmoles NO2− per 106 macrophages) is consistent with the small levels attributed to NOS3 activity in normal rat AMs (∼70 pmoles/106 cells (Miles et al., 1998)) and with the rate of NO release observed upon estrogen stimulation of human monocytes (Stefano et al., 1999). Figure 4 Download asset Open asset Estrogen-mediated activation of macrophage NOS3. Estrogen treatment of J774A.1 mouse or human U937 macrophages (A and B) increases killing of ingested pneumococci; this increased killing is prevented by the NOS inhibitors NLA or l-NMMA, but not control stereoisomers (n = 3–4, * = p < 0.01). (C) Western blot analysis shows >100-fold NOS3 in macrophages compared to the endothelial cell line bEnd.1; after 30 min, estrogen-treated (E2, estradiol, 0.2 ng/ml) J774A.1 mouse macrophages show increased phosphorylation of Akt and NOS 3, while normal female AMs show basally increased pAkt and pNOS3 compared to male AMs; (D) basal- and estrogen-enhanced phosphorylation of Akt and NOS3 are inhibited by wortmannin (Wm, 50 nM). (E) Inhibition of Akt with 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (10 µg/ml REF) prevents estrogen-mediated increased bacterial killing in J774A.1 cells (n = 3, * = p < 0.01). (F) Aerosol pre-treatment of male mice with albumin-conjugated estrogen 30 min before pneumococcal infection improves bacterial killing (n = 6, * = p < 0.01). (G) In ovariectomy-model of menopause, female mice lose their greater resistance to pneumococcal pneumonia after 10 weeks, an effect reversed by treatment with estrogen prior to infection, n > 8 for control, 10 week groups; n = 3 for 2 and 5 week groups; * = p < 0.01. https://doi.org/10.7554/eLife.03711.007 Studies of NOS3 in endothelial cell biology have identified rapid activation via phosphorylation of NOS3 at serine 1177 (Gonzalez et al., 2002) and have shown that estrogen mediates this activation via specific signaling cascades, such as phospho-inositol-3 kinase and protein kinase B, also known as Akt, (Chambliss and Shaul, 2002; Duckles and Miller, 2010). To investigate the pathway in macrophages, we cultured J774A.1 macrophages in hormone-free serum. Western blot analysis of responses after addition of estrogen to J774A.1 cells showed rapid phosphorylation of Akt and NOS3 in response to estrogen (Figure 4C; fold-increase 1.7 + 0.3, 1.7 + 0.1, respectively, n = 4–5, p < 0.01) and basally increased levels of pNOS3 and pAkt in normal female mouse AMs compared to male AMs (Figure 4C, fold-increase 1.7 + 0.4, 1.5 + 0.2, pAkt and pNOS3 respectively, n = 2–3, p < 0.01). The Western blot analysis also shows that the amount of NOS3 in the macrophage cell line and primary AMs is quite low, at least two orders of magnitude lower than present in the positive control bEnd.1 endothelial cell line samples. A role for PI3K in the signaling cascade was supported by the decrease in pAkt levels, either basally or after E2-treatment, in J774A.1 macrophages treated with the inhibitor wortmannin (Figure 4D; fold increase 2.3 + 0.2, 1.04 + 0.5, pAkt after E2 or E2 + wortmannin, respectively, n = 2–3, p < 0.05). To test this pathway more directly, we also used the Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (Takeuchi and Ito, 2004), to test whether it would block macrophage killing of pneumococci. This agent did not alter bacterial binding or internalization (not shown). As illustrated in Figure 4E, Akt inhibition completely abrogated estrogen-mediated enhanced killing, supporting a functional role for Akt in estrogen-mediated activation of macrophages. To begin testing of the translational potential of targeting NOS3, we first tested whether local delivery of estrogen to the lungs would improve resistance to bacterial pneumonia. We found that aerosol treatment of male mice before challenge with pneumococci improved the clearance of bacteria (Figure 4F). This was especially true if we used an estradiol conjugated to albumin, which acts to slow absorption of this lipophilic hormone (systemic uptake being the basis for a form of aerosolized estrogen-replacement therapy formerly used in menopausal women [Studd et al., 1999]). Populations at risk for localized outbreaks of primary pneumococcal pneumonia include elderly (post-menopausal) nursing home residents (Muder, 1998). We evaluated bacterial clearance in a mouse model of menopause that gradually develops several weeks after surgical ovariectomy (Chakraborty and Gore, 2004). We found a gradual decline in bacterial clearance in ovariectomized female mice to normal male levels at 10 weeks after ovariectomy; aerosolized estrogen treatment of ovariectomized mice reversed this decline and restored the normal, superior female clearance capacity (Figure 4G). Targeting NOS3 in primary pneumococcal pneumonia Statins can increase levels of NOS3 (Forstermann and Li, 2011) and have been associated with beneficial effects on incidence of hospitalization for pneumonia and subsequent mortality (Thomsen et al., 2008; Nielsen et al., 2012). To study possible effects on innate immunity and initial resistance to pneumonia, we measured the effects of statin therapy and estrogen-replacement therapy on the incidence of hospitalization with pneumonia in a large well-defined female human population in Denmark between 1997 and 2012. Table 1 shows the characteristics of 28,576 female subjects with first-time hospitalized pneumonia and 142,880 age-matched female population control subjects. The unadjusted OR results in Table 2 indicate that in crude analyses, estrogen users had a similar pneumonia hospitalization risk than estrogen non-users, and statin users had a slightly higher pneumonia risk than statin non-users. After controlling for comorbidity and other confounding factors associated with drug use, we found that receiving statin therapy reduced incidence of pneumonia requiring hospitalization in females. In addition, estrogen-replacement therapy was associated with a similar reduction, and women receiving both statin and estrogen therapy showed an even greater reduction in pneumonia risk (adjusted OR Table 1 of 28,576 female subjects with first-time hospitalized pneumonia and 142,880 age-matched female population control subjects from Denmark, at time of pneumonia comorbidity low (Mizgerd, 2006; Shrestha et al., high use use use use Statins and estrogen, use Statins and estrogen, no or Table 2 for first-time hospitalized pneumonia associated with use of statins estrogen and estrogen + statins in (n = population controls (n = OR OR use of statins or use of statins before use of estrogen before use of both statins and estrogen before * for and hospitalization for of comorbidity before and Table 1). To investigate statin effects on macrophage with bacteria, we first found that in vitro treatment of macrophages for hr with to increased levels of NOS3 and its (Figure fold-increase + 0.2, 1.7 + NOS3, 3 hr after respectively, n = 2–3, p < 0.05). This was associated with killing of internalized bacteria (Figure We measured in vivo clearance of pneumococci in mice treated with statins and found improved clearance in both genders, but this was not seen when mice deficient in NOS3 were used (Figure mice treated with statins showed improved survival with pneumococcal pneumonia (Figure Figure 5 Download asset Open asset Statins enhance innate immune resistance to S. pneumoniae via NOS3. (A) In vitro treatment of J774A.1 mouse macrophages with increases levels of pNOS3 and NOS3 and (B) increases killing of internalized bacteria (n = * = p < 0.01). (C) In vivo, pre-treatment of mice with significantly improves bacterial clearance in wild-type mice (n = 8, * = p < 0.01 vs male = p < 0.01 vs males + but has no effect on either male or female mice. (D) male mice with pneumococcal pneumonia show improved survival (n = 8, * = p < 0.01). (E) a small of NOS3, increases bacterial killing by mouse macrophages in vitro (n = 3, * = p < 0.01) (F) of male mice with by either subcutaneous or improves in vivo bacterial clearance, an effect not seen in male mice (n = * = p < 0.01). is a small-molecule compound that increases NOS3 and protein levels by mechanisms from statins by and may a more therapeutic et al., 2008). We found that treatment improved bacterial killing by macrophages in vitro (Figure and that mice treated with either by or subcutaneous showed increased clearance of pneumococci in vivo, an effect not observed in Nos3−/− mice (Figure Targeting NOS3 in secondary pneumococcal pneumonia pneumococcal pneumonia remains a major problem after primary influenza et al., 2013) and can be by mice with a of influenza and the lungs with pneumococci This is a time of susceptibility to secondary infection (Sun and Metzger, 2008; Ghoneim and McCullers, as illustrated by the low of pneumococci needed to cause pneumonia compared to the used in the primary pneumonia We evaluated the pharmacologic agents that in primary pneumonia model for potential to reduce susceptibility or of secondary pneumonia after influenza. We found that treatment of mice with statins improved survival in a (Figure We observed similar improved survival in mice treated with (Figure and also found this treatment improved bacterial clearance measured at 24 hr after challenge (Figure Figure Download asset Open asset Statins and improve survival from post-influenza secondary pneumococcal pneumonia. mice were to from influenza 1 and challenged with S. pneumoniae with (A) or or (B) caused a in survival (n = * = p < 0.01). (C) treatment also lead to improved bacterial clearance 24 hr after pneumococcal challenge in this post-influenza model (n = 6, * = p < 0.01). Discussion We sought to identify new approaches to innate immunity to bacterial pneumonia by the basis for gender differences in resistance to pneumococcal pneumonia. We that estrogen mediates greater host resistance to pneumonia in female mice via effects on the constitutively expressed NOS3 in lung macrophages. The basis for this data in mouse cells or models using pharmacologic and genetically deficient A similar advantage is seen in female human primary AMs and estrogen-treated human macrophages. identify a novel host defense mechanism and a function for the main NOS found in normal human macrophages. Pharmacologic targeting of NOS3 with one type of drug already in clinical use and another small-molecule lead compound (AVE3085) to improved bacterial clearance and improved survival from secondary pneumococcal pneumonia. The by which macrophage NOS3 to improved killing of ingested bacteria include either or in with superoxide 2004), but NOS3 might also to mediate signaling et al., 2013). The very low of NO by macrophage NOS3 the that mechanisms to its contribution to bacterial may include increased of NOS3 after estrogen-mediated phosphorylation to of NOS3 production of NO bacteria by and of NO with