The adipocyte-derived hormone leptin regulates energy homeostasis and the innate immune response. We previously reported that leptin plays a protective role in bacterial pneumonia, but the mechanisms by which leptin regulates host defense remain poorly understood. Leptin binding to its receptor, LepRb, activates multiple intracellular signaling pathways, including ERK1/2, STAT5, and STAT3. In this study, we compared the responses of wild-type and s/s mice, which possess a mutant LepRb that prevents leptin-induced STAT3 activation, to determine the role of this signaling pathway in pneumococcal pneumonia. Compared with wild-type animals, s/s mice exhibited greater survival and enhanced pulmonary bacterial clearance after an intratracheal challenge with Streptococcus pneumoniae. We also observed enhanced phagocytosis and killing of S. pneumoniae in vitro in alveolar macrophages (AMs) obtained from s/s mice. Notably, the improved host defense and AM antibacterial effector functions in s/s mice were associated with increased cysteinyl-leukotriene production in vivo and in AMs in vitro. Augmentation of phagocytosis in AMs from s/s mice could be blocked using a pharmacologic cysteinyl-leukotriene receptor antagonist. Phosphorylation of ERK1/2 and cytosolic phospholipase A2 α, known to enhance the release of arachidonic acid for subsequent conversion to leukotrienes, was also increased in AMs from s/s mice stimulated with S. pneumoniae in vitro. These data indicate that ablation of LepRb-mediated STAT3 signaling and the associated augmentation of ERK1/2, cytosolic phospholipase A2 α, and cysteinyl-leukotriene synthesis confers resistance to s/s mice during pneumococcal pneumonia. These data provide novel insights into the intracellular signaling events by which leptin contributes to host defense against bacterial pneumonia.
The adipocyte-derived hormone leptin, first described as a satiety signal whose synthesis correlates with total body fat mass (1), plays an essential role in regulating energy homeostasis and innate and adaptive immunity (2). We and others have reported that blood and tissue leptin levels increase during bacterial infections and that this adipocytokine plays an essential role in the host defense against pulmonary infections (3–6). Both genetically leptin-deficient ob/ob mice and mice rendered leptin-deficient by fasting exhibit impaired pulmonary bacterial clearance and enhanced lethality during bacterial pneumonia (3, 4, 7). The provision of exogenous leptin reconstituted host defense in fasted animals and improved these responses in ob/ob mice in vivo (4, 7).
Associated with the defects in pulmonary host defense in vivo, we also observed impairments in phagocytosis and killing of bacteria in alveolar macrophages (AMs) and polymorphonuclear neutrophils (PMNs) from leptin-deficient animals that could be restored with exogenous leptin in vitro (3, 4, 7, 8). The mechanisms by which leptin regulates leukocyte antimicrobial functions include a direct effect mediated through signal transduction cascades initiated by the leptin receptor (LepR) leading to the activation of the respiratory burst (9) and cytoskeletal rearrangement for chemotactic responsiveness (10) and an indirect effect induced by the ability of leptin to enhance proinflammatory mediator synthesis in these cells. Leptin primes macrophages, and other leukocytes, for enhanced cytokine synthesis (11, 12), reactive oxygen intermediate and NO production (9, 13, 14), and adhesion receptor expression (8, 15).
In particular, we have previously observed reduced leukotriene synthesis in AMs obtained from mice rendered leptin deficient by genetic means (ob/ob mice) or by energy malnutrition (3, 7). In both cases, the provision of exogenous leptin restored leukotriene synthetic capacity and bacterial phagocytosis in cells from these animals. We have also reported that exogenous leptin enhances the ability of AMs obtained from wild-type animals to release arachidonic acid for subsequent conversion to leukotrienes (LTs) and PGE2 (16). The underlying mechanism for this enhancement was an increase in cytosolic phospholipase A2 γ expression.
The ability of leptin to stimulate macrophage innate immune responses requires the so-called long isoform of its receptor (LepRb) (2). Leptin binding to LepRb activates the receptor-associated Jak2, a tyrosine kinase that phosphorylates tyrosine residues 985, 1077, and 1138 of LepRb (Fig. 1A). LepRb–Jak2 signaling fails to mediate most of leptin’s actions in vivo, suggesting important roles for the signaling pathways controlled by LepRb tyrosine phosphorylation (17). LepRb Tyr985 recruits SH2-containing tyrosine phosphatase and growth factor binding 2, which promotes the activation of the ERK1/2 MAPK pathway (18). LepRb Tyr1077 mediates the activation of STAT5 (19), and Tyr1138 mediates the phosphorylation-dependent activation of STAT3. Importantly, in addition to its other actions, Tyr1138-mediated STAT3 signaling promotes the transcription of the suppressor of cytokine signaling 3 (SOCS3), an inhibitor of Jak2 and ERK activation known to attenuate LepRb signaling (20, 21). Leptin signaling via LepRbS1138 (containing a substitution mutant for Tyr1138 and thus null for STAT3 signaling) fails to promote SOCS3 accumulation and thus demonstrates augmented leptin-stimulated Jak2 and ERK1/2 signaling (Fig. 1B) (20). SOCS3 also inhibits signaling events mediated by gp130-related cytokine receptors such as the IL-6R (22).
LepRb-mediated STAT3 signaling (LepRb→STAT3) is very important for many aspects of leptin action in vivo, as mice expressing a “knocked-in” mutant LepRbS1138 (Leprs1138/s1138; s/s mice) from within the endogenous Lepr locus fail to mediate leptin-stimulated STAT3 signaling and are hyperphagic, obese, and insulin resistant, in addition to exhibiting increased circulating corticosterone levels similar to LepRb-null db/db mice (23, 24).
The role of LepRb→STAT3 signaling during an inflammatory response plays an important role in experimentally induced intestinal and hepatic inflammation (25) but not arterial neointima formation after vascular injury (26). In this study, we assessed the role of LepRb→STAT3 signaling in the innate immune response against bacterial infection of the lung by comparing the responses of wild-type and s/s mice in a murine model of pneumococcal pneumonia.
Materials and Methods
Heterozygous C57BL/6 (back-crossed for eight generations or greater) Leprs1138/+ mice were intercrossed in the University of Michigan Unit for Laboratory Animal Medicine (Ann Arbor, MI) to generate age- and gender-matched male and female (Leprs1138/s1138) s/s and wild-type (+/+; WT) littermates 8 to 14 wk of age (23). Animals were genotyped by TaqMan single nucleotide polymorphism allelic discrimination assay. Animals were treated according to National Institutes of Health guidelines for the use of experimental animals with the approval of the University of Michigan Committee for the Use and Care of Animals.
Immunohistochemical staining and image acquisition of lung sections
Lungs were removed from WT and s/s mice, inflated with 0.5 ml PBS or PBS with 1 μg/ml murine leptin (EMD Biosciences, La Jolla, CA) through the trachea, and suspended in PBS at 37°C. After 5, 15, and 30 min, the lungs were completely filled with and immersed in 4% paraformaldehyde to fix the lung. As previously described (27), fixed tissues were embedded in paraffin, sectioned, mounted, dewaxed in Americlear (Stephens Scientific, Richard-Allan, Riverdale, NJ), and rehydrated through decreasing concentrations of ethanol. Sections were quenched of endogenous peroxidase activity by treatment with 0.3% hydrogen peroxide for 30 min, washed, and blocked with Powerblock (InnoGenex, San Ramon, CA). Rabbit polyclonal Ab against p-STAT3 (Cell Signaling Technology, Danvers, MA) was prepared in PBS containing 0.1% BSA (titer 1:750) and applied overnight at 4°C. After washing with 0.1% BSA in PBS, slides were probed with secondary Ab (biotinylated goat anti-rabbit, 1:250) for 30 min at 37°C. After an additional washing step, sections were treated with avidin peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) for 30 min at room temperature. 3, 3′ Diaminobenzidine was used as the peroxidase substrate, and preparations were counterstained with Harris’s hematoxylin. Bright-field images were acquired using a Nikon Eclipse 50i microscope, Nikon DS-5M camera, and NIS-Elements BR 3.0 software.
Quantitative PCR for SOCS3
Briefly, RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) and converted to cDNA using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). cDNA was analyzed in triplicate via quantitative RT-PCR for GAPDH and SOCS3 gene expression (as supplied by Applied Biosystems) using an Applied Biosystems 7500 real-time thermal cycler. Gene expression was normalized to GAPDH expression, and relative mRNA expression was calculated via the 2−ΔΔCt method.
S. pneumoniae infection
S. pneumoniae serotype 3, ATCC 6303 (American Type Culture Collection, Manassas, VA), was grown in Todd Hewitt Broth Media (BD, Sparks, MD) at 37°C in 5% CO2 to midlog phase. The bacteria were pelleted by centrifugation (16,000 × g for 5 min), washed twice in endotoxin-free PBS (Invitrogen), and the concentration of bacteria was determined spectrophotometrically (A600) and confirmed by plating serially diluted bacteria on soy-based blood agar plates (Difco, Detroit, MI). The virulence of this organism was maintained by culturing bacteria obtained from the spleens of mice rendered bacteremic 24 h after an intratracheal (i.t.) challenge with 106 CFU S. pneumoniae. Mice were anesthetized as previously described using ketamine and xylazine (28), a small incision was made to expose the trachea, and the bacterial inoculum (5 × 104 CFU suspended in 30 μl PBS) was injected using a 26-gauge needle via the i.t. route. The incision was closed using 3M Vetbond (3M Animal Care Products, St. Paul, MN), and mice were aided in their recovery with a heating pad.
Determination of survival, bacterial loads, cytokines, and cysteinyl-leukotrienes
After i.t. inoculation with S. pneumoniae, mice were evaluated for survival every day for 10 d. In a separate group of mice, lungs and spleens were obtained from euthanized mice 24 and 48 h postinfection, homogenized, serially diluted, and plated on blood agar plates for determining bacterial burdens. Cytokine (CXCL2 [MIP-2], IL-6, IL-10, IL-12, and TNF-α) and leptin levels in whole-lung homogenates were determined using commercially available enzyme immunoassay (EIA) kits (murine leptin, EMD Biosciences; murine cytokines, R&D Duoset, R&D Systems, Minneapolis, MN). Lung homogenate cysteinyl-leukotriene (cysLT) levels were determined using an EIA kit according to the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI).
Lung leukocyte enumeration
Twenty-four and forty-eight hours postinfection, lung leukocytes were obtained from mice by bronchoalveolar lavage (BAL) after CO2 asphyxiation, and differential counts were performed on cells after staining with a modified Wright-Giemsa stain (American Scientific Products, McGraw Park, IL).
Isolation and culture of AMs
Resident AMs were recovered from mice by lung lavage as previously described (29), resuspended in RPMI 1640 (Life Technologies, Invitrogen, Carlsbad, CA) to a concentration of 2 × 106 cells per milliliter, and allowed to adhere to tissue culture plates for 1 h (37°C, 5% CO2). To stimulate AMs for LT and PGE2 production, the cells were cultured with and without heat-killed S. pneumoniae (multiplicity of infection [MOI] 1000:1) for 4 h. Cell culture medium was recovered and stored at −70°C until assays were performed to determine the levels of cysLTs, leukotriene B4 (LTB4), and PGE2 using EIA kits according to the manufacturer’s instructions (Cayman Chemical).
Measurement of reactive oxygen intermediate production
AMs were adhered to 384-well plates at a concentration of 1.25 × 105 cells/well and cultured overnight in RPMI 1640 containing 10% FCS and antibiotics. On the next day, the medium was replaced with PBS containing 10 μM 2′,7′-dichlorodihydrofluorescein, and the cells were cultured for 1 h. The medium was then replaced with warmed HBSS, and the cells were stimulated with heat-killed S. pneumoniae using an MOI of 50:1. Reactive oxygen intermediate production was assessed every 30 min for 2.5 h by measuring fluorescence using a Spectramax Gemini XS fluorometer (Molecular Devices, Sunnyvale, CA) with excitation/emission setting at 493/522 nm.
AMs were adhered to 96-well plates at a concentration of 2 × 105 cells/well and cultured with DMEM supplemented with 1% sodium pyruvate (Invitrogen) containing 10% FCS and penicillin/streptomycin with or without 10 μg/ml lipoteichoic acid from Staphylococcus aureus (Sigma-Aldrich) and 10 ng/ml IFN-γ (R&D Systems) for 24 h. NO production was determined by measuring stable nitrite (NO2–) concentrations using a modified Griess reaction with a commercially available assay kit according to the manufacturer’s instructions (Cayman Chemical).
Fluorometric assay of AM phagocytosis
AM phagocytosis of S. pneumoniae was assessed using a previously published protocol for determining the ingestion of fluorescent, FITC-labeled, heat-killed S. pneumoniae serotype 3 (FITC–S. pneumoniae). Murine AMs (1.25 × 105), obtained from the BAL fluid of age-matched WT and s/s female mice, were adhered and seeded in replicates of eight to 384-well tissue culture plates with opaque sides and optically clear bottoms (Costar; Corning Life Sciences, Lowell, MA) and cultured overnight with RPMI 1640 with 5% penicillin/streptomycin and 10% FCS (Invitrogen). On the following day, FITC–S. pneumoniae were opsonized with 10% normal rat-derived nonimmune serum. AMs pretreated with RPMI 1640 medium alone, LTB4 receptor antagonist (CP105, 696; 2 μM), or the cysLT receptor antagonist (MK571; 1 μM; Cayman Chemical) for 15 min were then infected with FITC–S. pneumoniae using an MOI of 150:1 for 60 min to allow phagocytosis to occur. Trypan blue (250 μg/ml; Molecular Probes) was added for 1 min to quench the fluorescence of extracellular bacteria, and fluorescence was determined using a SpectraMax Gemini EM fluorometer 485-nm excitation/535-nm emission (Molecular Devices, Sunnyvale, CA). The phagocytic index was calculated as previously described in relative fluorescence units (28, 30). Three separate experiments were conducted with eight replicate wells for every experimental condition, and the relative fluorescence units were normalized to the control condition (untreated AMs from WT animals) in each different experiment.
Tetrazolium dye reduction assay of bacterial killing
The ability of bacteria to survive within the AM was quantified using a tetrazolium dye reduction assay, as previously described (28). Briefly, 2 × 106/ml mouse AMs, prepared as described previously, were seeded in duplicate 96-well tissue culture dishes (31). The next day, S. pneumoniae were opsonized with 10% normal serum, as previously described (32). Cells were then infected with an 0.1-ml suspension of opsonized S. pneumoniae (2 × 107 CFU/ml, MOI of 50:1) for 120 min to allow phagocytosis to occur. The bacterial killing protocol was assessed as described elsewhere (32, 33). The intensity of the A595 was directly proportional to the number of intracellular bacteria associated with the macrophages (32). Results were expressed as percentage of survival of ingested bacteria, where the survival of ingested bacteria = 100% × A595 control (phagocytosis) plate/A595 experimental (phagocytosis + killing) plate.
Immunoblot analysis for ERK1/2, p-ERK1/2, cytosolic phospholipase A2 α (cPLA2α), p-cPLA2α, STAT3, and p-STAT3
AMs were obtained from either WT or s/s mice, plated 4 × 106 cells per well, and cultured overnight in RPMI 1640 with BSA. On the following day, the cells were cultured with medium alone or leptin (1 μg/ml) for 5, 15, or 30 min. In some experiments, AMs were pretreated with medium alone or with the ERK1/2 inhibitor UO126 (1 μM) for 15 min and stimulated with heat-killed S. pneumoniae for 30 min. The macrophages were then washed with HBSS and scraped with ice-cold lysis buffer (RIPA buffer; Sigma), and cells were disrupted with sonication (10 bursts at 20% duty/cycle). Twenty micrograms of protein, as determined by a modified Coomassie blue binding assay (Pierce Chemical, Rockford, IL), was separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. Membranes were probed with the rabbit polyclonal Abs against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:2000), p44/42 MAPK (ERK1/2) (1:2000), cPLA2α (1:1000), phospho (Ser505)-cPLA2α (p-cPLA2α) (1:1000), STAT3 (1:1000), or p-STAT3 (1:1000) (Cell Signaling Technology). Primary Abs were detected using HRP-conjugated goat anti-rabbit secondary Ab (titer 1:5000) and visualized with the ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ). The densities of the luminescent bands were quantitated in appropriately exposed nitrocellulose by using Image Reader (FujiFilm). The densities of the luminescent bands were quantitated in appropriately exposed nitrocellulose using Adobe Image Reader. The density value of the p-cPLA2α and p-ERK1/2 blots were divided by the density value of the cPLA2α and ERK1/2 blots, respectively, to normalize the relative band densities.
Reduced levels of SOCS3 mRNA and ablation of STAT3 signaling in AMs from s/s mice
To determine if STAT3 signaling plays an important role in the ability of leptin to regulate pulmonary antibacterial host defense, we first confirmed that this pathway was ablated in s/s mice. After culturing AMs with leptin for 5 min, immunoblot analysis of cell lysates revealed STAT3 staining in cells from both animals. However, p-STAT3 staining was found in AMs from WT but not s/s mice cultured with leptin (Fig. 1C). We did not observe p-STAT3 staining in AMs from either WT or s/s mice after culturing AMs with leptin for 15 or 30 min (data not shown). Scheller et al. (34) also observed a rapid phosphorylation of STAT3 in bone marrow stromal cells that peaked at 5 min and was absent 30 min after the addition of leptin to the cell culture medium. As shown in Fig. 1D, SOCS3 mRNA levels were ∼4-fold lower in AMs from s/s mice compared with those from WT animals. Next, we inflated the lungs obtained from WT and s/s mice with warm PBS with or without leptin (1 μg/ml) ex vivo for 5, 15, and 30 min prior to fixation and evaluated tissue sections stained for p-STAT3 (Fig. 2). We observed p-STAT3 staining in AMs and, to a lesser extent, in the alveolar wall in the lungs of WT animals, as indicated by brown staining, 15 and 30 min after inflating the lungs with PBS containing leptin (Fig. 2C, 2D). In contrast, we did not observe p-STAT3 staining in lung sections prepared from s/s mice at any time point (Fig. 2E–H). These results indicate that SOCS3 levels are substantially reduced in AMs from s/s mice. Leptin initiates STAT3 activation in AMs and in alveolar epithelial cells, in vitro and in situ, and these responses are absent in s/s mice.
Improved survival, enhanced pulmonary bacterial clearance, and reduced bacterial dissemination in s/s mice after i.t. challenge with S. pneumoniae
We have previously observed that leptin deficiency induced by genetic means (ob/ob mice) (3, 4) or energy deprivation (7) impairs pulmonary host defense against bacterial pneumonia. To determine the role for LepRb→STAT3 signaling in pulmonary host response against bacterial pneumonia, we infected WT and s/s mice via the i.t. route with S. pneumoniae. As shown in Fig. 3A, s/s mice experienced significantly lower mortality (36%) compared with that of WT mice (65%, p < 0.05) 10 d after pneumococcal challenge. There were no additional deaths 10 d after the infection. Because the increased survival observed in the s/s mice might indicate enhanced clearance of S. pneumoniae from the lungs, we assessed lung and spleen bacterial burdens 24 and 48 h after S. pneumoniae challenge. As shown in Fig. 3B, lung bacterial loads were 1- and 2-log fold lower in s/s mice 24 and 48 h, respectively, after S. pneumoniae challenge. Likewise, spleen bacterial burdens were also reduced at these time points in s/s mice (although this trend was not significant at the 24-h time point [p = 0.06]). These results suggest that ablation of LepRb→ STAT3 signaling enhances pulmonary host defense against pneumococcal pneumonia by improving bacterial clearance.
Minor differences between WT and s/s mice in leukocyte recruitment after S. pneumoniae challenge
The influx of leukocytes to the lungs during bacterial pneumonia is critically important for the clearance of bacteria from the lung and to prevent dissemination to the peripheral circulation. We have previously reported increased PMN recruitment to the lungs of leptin-deficient (ob/ob) mice and attenuated leukocyte recruitment in mice rendered leptin deficient by fasting in response to pneumococcal pneumonia (4, 7). To determine whether the ablation of LepRb→STAT3 signaling might increase leukocyte recruitment to the lung to promote increased resistance to pneumococcal pneumonia, we collected inflammatory cells from the BAL fluid of WT and s/s mice 24 and 48 h postinfection. Although the number of PMNs recovered from WT mice 24 h after S. pneumoniae challenge was modestly elevated compared with that of s/s mice, there were no other differences in leukocyte counts (Fig. 4). However, we did observe a greater percentage of monocytes/macrophages in s/s mice and a higher percentage of PMNs in WT mice 24 h postinfection (Fig. 4C, 4D). This result indicates that LepRb→STAT3 signaling during pneumococcal pneumonia does not play a significant role in pulmonary leukocyte recruitment and that enhanced leukocyte effector function must underlie the augmentation of pulmonary bacterial clearance observed in s/s animals.
Increased pulmonary cysLT and delayed cytokine production in s/s mice during pneumococcal pneumonia
Leptin deficiency has been shown to affect the production of cytokines and LTs in animals during bacterial pneumonia and mycobacterial infections of the lung (4–7). Although the influence of LepRb→STAT3 signaling on cytokine production during bacterial infection has not been evaluated, a recent report by Gove et al. (25) revealed a reduction in cytokine production in s/s mice after chemically induced colitis and hepatitis. We evaluated the production of cytokines (IL-6, IL-10, IL-12, MIP-2, and TNF-α), cysLTs, and leptin in the lungs of WT and s/s mice after i.t. S. pneumoniae challenge to determine the impact of LepRb→STAT3 signaling on these responses. Consistent with obesity and known hyperleptinemia, lung homogenate leptin levels were elevated in s/s mice at both time points during pneumococcal pneumonia. Compared with their WT counterparts, in s/s mice the levels of IL-6, IL-10, IL-12, MIP-2 (not significantly), and TNF-α were reduced 24 h after S. pneumoniae challenge. There were no differences in lung homogenate cytokines 48 h postinfection suggesting that differences in these cytokines cannot explain the improved resistance of s/s mice to pneumococcal pneumonia. In contrast, the levels of cysLTs in the lungs of s/s mice were elevated relative to those in WT animals by ∼300 and 70% 24 and 48 h, respectively, after S. pneumoniae infection. These results suggest that ablation of LepRb→STAT3 signaling enhances the production of LTs in s/s mice during pneumococcal pneumonia (Fig. 5).
Improved phagocytosis and killing of S. pneumoniae and enhanced eicosanoid synthesis in vitro by AMs from s/s mice
We were intrigued by the enhancement of LTs in s/s mice during pneumococcal pneumonia in vivo because we have previously reported that exogenously administered LTs can enhance bacterial phagocytosis in leukocytes in vitro and improve pulmonary bacterial clearance of S. pneumoniae in vivo (31, 35). To determine the relevance of increased LT synthetic capacity to the improved pulmonary bacterial clearance observed in s/s mice, we assessed the ability of AMs obtained from WT and s/s mice to phagocytose and kill S. pneumoniae in vitro. As shown in Fig. 6A, the capacity of AMs from s/s mice to phagocytose serum opsonized FITC–S. pneumoniae was nearly 2-fold greater than that of WT AMs. We also observed a modest improvement in the ability of AMs from s/s mice to kill ingested S. pneumoniae (WT 86 ± 3.8% and s/s 75 ± 6.7% survival of ingested S. pneumoniae, n = 5 separate experiments, p < 0.05 using a Student t test). This enhancement in bacterial killing was associated with a slight increase in reactive oxygen intermediate production in AMs stimulated with heat-killed S. pneumoniae for 90, 120, and 150 min (data not shown) (p < 0.05 by ANOVA) but not NO synthesis. Next, we examined eicosanoid synthesis in AMs obtained from s/s mice stimulated with heat-killed S. pneumoniae. As shown in Fig. 6B, we found that cells from s/s mice produced 200–250% more LTB4, cysLTs, and PGE2 than that of WT mice. In contrast, we did not observe any difference between WT and s/s mice in the ability of AMs to produce cytokines (IL-6, IL-12, IL-10, MIP-2, TNF-α) in vitro (data not shown). To determine if the increase in phagocytosis of S. pneumoniae in AMs from s/s mice was dependent on the observed increase in LT synthesis, cells were pretreated with the LTB4 receptor (BLT1) antagonist CP105,696 and CysLT receptor (CysLT1) antagonist MK571. Although blocking BLT1 attenuated phagocytosis in AMs from WT mice, it did not significantly affect the phagocytic capacity of cells from s/s mice (Fig. 6C). In contrast, the cysLT1 inhibitor attenuated bacterial phagocytosis in AMs from s/s mice to the level observed in cells from WT animals. Collectively, these data are consistent with a role for increased cysLT synthesis in the improved phagocytic capacity of AMs from s/s mice.
Increased ERK1/2 and cPLA2α activation in AMs from s/s mice
To determine the mechanism responsible for the increased production of both LTs and PGE2 observed in AMs stimulated with heat-killed S. pneumoniae, we first considered the possibility that other signal transduction pathways initiated by LepRb may be upregulated in cells from s/s mice. For example, LepRb→STAT3 signaling induces SOCS3 transcription, which attenuates LepRb/Jak2-mediated intracellular signaling (20, 21), and the ablation of LepRb→STAT3 signaling prevents SOCS3-mediated feedback inhibition of LepRb. The reduction in SOCS3 might enhance other signaling pathways, including ERK1/2. Potential mechanisms by which eicosanoid synthesis may be enhanced in cells from s/s mice might be through the increased expression of the PLA2 enzymes known to liberate arachidonic acid from membrane phospholipids for subsequent conversion to the eicosanoids. To address these possibilities, we stimulated AMs from WT and s/s mice with heat-killed S. pneumoniae in vitro and assessed the expression of total ERK1/2 and p-ERK1/2. As shown in Fig. 7A, immunoblotting of cell lysates prepared from AMs stimulated with heat-killed S. pneumoniae for 30 min revealed an enhancement of ERK1/2 activation as indicated by an approximate 5-fold increase in p-ERK1/2/total ERK blot density in cells from s/s mice compared with that of WT. Next, we evaluated the expression of cPLA2α and secretory phospholipase A2, enzymes that are known to liberate arachidonic acid from tissue phospholipids in response to bacterial stimulation for subsequent conversion to eicosanoids. While immunoblot analysis did not reveal differences in the expression of cPLA2α or secretory phospholipase A2 in AMs from WT and s/s mice (data not shown), we did observe an increase in the phosphorylation of cPLA2α (p-cPLA2α/total cPLA2α) in cells from s/s mice stimulated with heat-killed S. pneumoniae (Fig. 7B). In addition, when AMs were pretreated with the selective ERK/1/2 inhibitor UO126 prior to stimulation, we observed that cPLA2α phosphorylation was inhibited in AMs from both WT and s/s mice. UO126 also attenuated the enhanced cysLT synthetic capacity in AMs from s/s mice (Fig. 7C). This finding suggests that the enhanced eicosanoid biosynthesis in AMs obtained from s/s mice was due to an increase in p-ERK1/2–mediated cPLA2α phosphorylation, a mechanism that has been observed previously in human bone marrow-derived cells stimulated with leptin (36). These results suggest that the increased production of both LTs and PGE2 was due to the increased liberation of arachidonic acid via cPLA2α activation.
Using ob/ob and fasted mice, we have previously demonstrated that leptin deficiency is associated with impaired pulmonary host defense against bacterial pneumonia (3, 4, 7). In the current study, we assessed the role of LepRb-induced STAT3 signaling pathway in pneumococcal pneumonia by comparing the responses of s/s mice with their WT counterparts. Despite obesity, hyperglycemia, and glucocorticoid excess in s/s mice, we found that the lack of LepRb→ STAT3 signaling was protective during pneumococcal pneumonia, improving survival, pulmonary bacterial clearance, and LT synthesis in animals challenged with S. pneumoniae in vivo. The improvement in host defense in s/s mice was associated with a modest augmentation of bacterial killing in vitro, as well as enhanced AM phagocytosis of S. pneumoniae, which could be blocked with a cysLT receptor antagonist. We also observed an enhancement of ERK1/2 and cPLA2α phosphorylation in AMs from s/s mice, indicating the enhancement of other LepRb signal transduction cascades in these animals. These results reveal novel cellular mechanisms by which leptin regulates the innate immune response during bacterial pneumonia.
We chose not to include db/db mice in our studies for a number of reasons. First, bone marrow cells and neutrophils from C57BL/6J-m-db/db mouse (The Jackson Laboratory) do not produce superoxide due to the absence of p47phox, an essential component of the NADPH oxidase enzyme complex, in these cells (37). The respiratory burst initiated by NADPH oxidase plays an essential role in host defense against bacterial infections (38). Although some have observed greater susceptibility to bacterial infections in db/db mice, it is difficult to determine if this greater susceptibility is due to leptin receptor deficiency or a defect in the ability of leukocytes from these animals to produce superoxide (39, 40). Second, the respiratory burst also induces ERK1/2 activation in leukocytes, and the lack of this response in cells from db/db mice would complicate the interpretation of ERK1/2 signaling after stimulation with heat-killed S. pneumoniae (41). Finally, the inability of leptin to induce STAT3 phosphorylation in tissues of db/db mice has previously been reported (42).
Leptin mediates its effects in the lung by binding to short and long isoforms of LepR, whose expression has been identified in bronchial epithelial cells (43, 44) and alveolar type I and type II epithelial cells (45, 46). We have demonstrated previously that AMs obtained from ob/ob mice are responsive to leptin as phagocytosis and LT synthesis are enhanced after overnight culture with this adipocytokine (3, 16). In the current study, we report that leptin induces STAT3 activation in AMs, and to a lesser extent in alveolar epithelial cells, as indicated by p-STAT3 staining in lungs obtained from WT but not s/s mice inflated with PBS containing leptin ex vivo. This observation indicates AMs express the LepRb because it is the only isoform capable of inducing STAT3 signaling and identifies this cell type as a target for leptin’s actions in the lung (47).
The production of cytokines during the course of pneumococcal pneumonia plays an important role in enhancing the antimicrobial functions of resident AMs and leukocytes recruited to the lung (48, 49). Compared with WT mice, we observed reduced levels of cytokines (IL-6, IL-10, IL-12, MIP-2 [albeit not statistically significant] and TNF-α) and PMNs in the BAL fluid of s/s mice 24 h after S. pneumoniae challenge. This lower level of cytokine production and PMN recruitment in s/s mice was most likely due to the lower lung bacterial burdens compared with those of WT animals. The lower levels of cytokines and PMNs in the lungs of s/s mice also suggest more efficient pulmonary bacterial clearance by resident AMs, which are known to play an essential role in controlling pulmonary inflammation during pneumococcal pneumonia (50). Although PMNs are capable of killing S. pneumoniae (51), they can also cause collateral damage to the lung during pneumococcal pneumonia, which compromises host defense (50). The disparities in cytokines and lung leukocyte counts do not explain the differences between WT and s/s mice in pulmonary bacterial clearance or survival. However, we observed an enhancement of LT production in response to S. pneumoniae challenge, and this may have played a significant role in the enhancement of host defense in s/s mice. We have recently reported that 5-lipoxygenase knockout mice, which are LT deficient, are more susceptible to pneumococcal pneumonia and that the provision of exogenous LTB4 to the lungs via intranasal administration or aerosol improves pulmonary bacterial clearance in both WT and 5-lipoxygenase knockout mice in vivo (35). Other reports have also demonstrated that LTs play a protective role in pulmonary infections and they do so by augmenting the ability of leukocytes to phagocytose and kill microbes (31, 52–55).
In addition to greater levels of LTs in s/s mice, leptin levels were also elevated in the lungs of these animals after S. pneumoniae challenge. As adipose tissue is the principle source of leptin, it was not surprising that we found higher levels of this adipocytokine in lung homogenates of the obese s/s mice. We have previously reported that leptin exogenously administered to mice who were rendered leptin-deficient by means of genetic manipulation or energy deprivation improves the pulmonary clearance of S. pneumoniae (4, 7). Likewise, exogenous leptin administration has also been reported to restore Listeria monocytogenes clearance in ob/ob mice (39). Leptin is a pleotropic adipocytokine known to stimulate leukocytes directly for improved antimicrobial functions and indirectly by enhancing the synthesis of proinflammatory mediators such as the LTs (3, 9, 12, 16, 56).
Whereas increased leptin levels might have contributed to the improved response to pneumococcal infection in vivo, the finding that AMs from s/s mice demonstrated enhanced phagocytosis and cysLT production in vitro suggest that alterations in cell signaling, not circulating leptin levels, underlie the improved host defense against pneumococcal pneumonia in s/s mice. Furthermore, murine AMs do not produce significant quantities of leptin in vitro, and it is unlikely that the observed enhancement in phagocytosis and killing was the result of the AMs from s/s mice responding differently to leptin in culture due to altered LepRb intracellular signaling. These cells can synthesize copious amounts of LTs in response to S. pneumoniae as we have shown in Fig. 6. We have previously reported that cysLTs and LTB4 enhance AM phagocytosis of bacteria and that pharmacologic blockade of their synthesis or receptors attenuates this response (31). In the current study, we demonstrated that the enhanced phagocytosis in AMs from s/s mice could be blocked using a selective pharmacologic cysLT but not LTB4 receptor antagonist. This result suggests that endogenously produced cysLTs, whose synthesis was 10-fold greater than that of LTB4, enhanced phagocytosis in an autocrine manner. Although we did find that the LTB4 receptor antagonist attenuated phagocytosis in cells from WT animals, the overabundance of cysLTs produced by cells from s/s mice may have compensated for the pharmacologic blockade of the LTB4 receptor.
The observed enhancement of host defense in s/s mice suggests that the ability of leptin to augment the innate immune response occurs through intracellular signaling cascades that are independent of the LepRb-induced STAT3 pathway. This result does not, however, diminish the importance of STAT3 signaling during inflammation because we evaluated the deletion of LepRb→STAT3 in our studies but not the total deletion of this transcription factor. STAT3-mediated gene transcription of SOCS3 plays an important role in controlling inflammation in a negative feedback loop to inhibit cytokine (IL-6, IL-10, and IL-23) production (57). For example, targeted deletion of STAT3 in alveolar epithelial cells has been shown to exacerbate pulmonary edema and impair bacterial clearance in a murine model of Escherichia coli pneumonia (58). Similarly, ablation of STAT3 in endothelial cells or cells of the myeloid lineage (macrophages, dendritic cells, and PMNs) results in excessive cytokine production and inflammation in murine models of endotoxemia (59) and septic peritonitis (60). To determine whether the lack of LepRb-mediated STAT3 signaling in s/s mice may have enhanced other signals, we assessed the ability of heat-killed S. pneumoniae to induce ERK1/2 activation in AMs from WT and s/s mice in vitro. We observed increased pERK1/2 levels after stimulation in cells from s/s mice as determined by immunoblot analysis. This finding was surprising because these experiments were conducted in cell culture media in the absence of leptin. Although we did not observe increased cytokine production in either the lung homogenates of s/s mice after S. pneumoniae challenge in vivo or in AMs stimulated with heat-killed S. pneumoniae in vitro (data not shown), we did find an increase in cPLA2α phosphorylation, which is known to enhance the ability of this enzyme to liberate arachidonic acid from tissue phospholipids. We also demonstrated that the enhanced cPLA2α phosphorylation and increased LT synthesis observed in AMs from s/s mice stimulated with heat-killed S. pneumoniae could be blocked with a selective pharmacologic inhibitor of ERK1/2, UO126. Likewise, a report by Kim et al. (36) demonstrated that exogenous leptin enhanced arachidonic acid release in human bone marrow stromal cells and this response could be blocked by pretreating these cells with inhibitors of ERK1/2 (U0126 and PD98059) or the selective cPLA2α, inhibitor AACOCF3. Therefore, the increased ERK1/2 and cPLA2α phosphorylation observed in AMs from s/s mice provides a mechanism to explain the enhanced production of both LTs and PGE2, oxygenated metabolites of arachidonic acid, produced by AMs from s/s mice.
Although we have shown that the enhanced host responses of s/s mice against S. pneumoniae in vivo and in vitro may have been due to their increased LT synthetic capacity, it is possible that other mechanisms may have contributed to the observed improvements in host defense of these animals. The enhanced heat-killed S. pneumoniae-induced ERK1/2 activation in AMs from s/s mice was associated with reduced SOCS3 mRNA levels. SOCS3 is not only a negative regulator of LepRb but also inhibits gp130-related cytokine (such as IL-6 and IL-12R) receptor signaling (21, 22). It is possible that augmented responses to IL-6 and IL-12, due to reduced SOCS3 levels, may have also contributed to the observed improvements in pulmonary bacterial clearance in s/s mice.
In summary, we report the observation that s/s mice are protected against pneumococcal pneumonia. The improvement in pulmonary bacterial clearance and survival in s/s mice was associated with an augmentation of AM phagocytosis of S. pneumoniae in vitro that was dependent upon the observed enhancement of LT synthesis in cells from these animals. These effects appear to be mediated by intracellular signaling pathways that are independent of LepRb→STAT3. We have also provided a mechanism by which increased ERK1/2 activation enhanced cPLA2α activity for subsequent increases in LT synthetic capacity in cells from s/s mice. These results provide a crucial, previously unrecognized mechanism by which LepRb signaling contributes to host defense.
We thank Beth Randall and Nicholas Zlatarov, who helped perform some of the experiments, Justin Jones for assistance in breeding and genotyping experimental animals, and Erica Schellar for advice on immunoblot analysis of p-STAT3/STAT3.
Disclosures The authors have no financial conflicts of interest.
This work was supported by National Institutes of Health Grants HL077417 (to P.M.) and DK56731 (to M.G.M.).
Abbreviations used in this article:
cytosolic phospholipase A2 α
multiplicity of infection
suppressor of cytokine signaling 3