Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease associated with cigarette smoking. Alterations in local lung and systemic iron regulation are associated with disease progression and pathogenesis. Hepcidin, an iron regulatory peptide hormone, is altered in subjects with COPD; however, the molecular role of hepcidin in COPD pathogenesis remains to be determined. In this study, using a murine model of smoke-induced COPD, we demonstrate that lung and circulating hepcidin levels are inhibited by cigarette smoke. We show that cigarette smoke exposure increases erythropoietin and bone marrow–derived erythroferrone and leads to expanded but inefficient erythropoiesis in murine bone marrow and an increase in ferroportin on alveolar macrophages (AMs). AMs from smokers and subjects with COPD display increased expression of ferroportin as well as hepcidin. Notably, murine AMs exposed to smoke fail to increase hepcidin in response to Gram-negative or Gram-positive infection. Loss of hepcidin in vivo results in blunted functional responses of AMs and exaggerated responses to Streptococcus pneumoniae infection.

Chronic obstructive pulmonary disease (COPD) is an incurable, life-limiting inflammatory lung disease associated with smoking and the third leading cause of mortality worldwide (1). Although the overwhelming risk factor for the development of COPD is tobacco smoking, genetic susceptibility loci involving iron-associated genes have highlighted the role of abnormal iron metabolism in disease pathogenesis (26). Specifically, excessive intracellular iron loading in the lung exacerbates inflammation and the severity of disease progression in murine COPD models and in patients with COPD (711). Iron is mostly localized inside lung tissues with remaining metal associated with bronchoalveolar lavage (BAL) protein (12). In a similar manner to other organs, the lung most likely obtains its iron from the pulmonary vasculature. Such a supply of iron to the lung depends on its systemic plasma availability, which is controlled by the hepcidin/ferroportin regulatory axis (13). Hepcidin (encoded by HAMP), a small peptide hormone chiefly produced in the liver, regulates and is in turn regulated by systemic iron levels (14). Hepcidin expression and release by the liver is induced by increased serum iron, by bone morphogenic protein (BMP) signaling (15), and by a number of proinflammatory cytokines, including IL-6 (16). In general, upregulation of hepcidin expression results in the inhibition of cellular iron export and reduces the influx of iron into the plasma from stores, as well as blocking further absorption of dietary iron (17). Ferroportin (encoded by the SLC40A1 gene) is a membrane-bound transporter that serves as the major exporter of iron from mammalian cells, including macrophages that recycle iron (18). Hepcidin induces ferroportin endocytosis and subsequent lysosomal degradation (13), which in turn decreases iron efflux from exporting cells (13).

Hepcidin, originally described as a cationic antimicrobial peptide (19, 20), is also found in tissues with no recognized role in systemic iron homeostasis, including the heart (21), the brain (22), and the kidney (23). The function of this extrahepatic hepcidin remains unknown, but one hypothesis is that it is involved in local iron control (24). Both hepcidin and ferroportin have documented expression in the lung (13, 20, 25), and resistance of ferroportin to hepcidin binding, as well as hepcidin deficiency results in pulmonary iron accumulation (13, 20), suggesting the ferroportin/hepcidin axis plays a role in pulmonary iron regulation. Little is known about the production or role of hepcidin in lung disease, although emerging conflicting evidence suggests that the hepcidin/ferroportin axis may be disrupted in patients with COPD. Specifically, serum hepcidin is increased in patients with mild-to-moderate disease or during an exacerbation event (increase in respiratory symptoms that require medical intervention) (26, 27) but is decreased in severe end-stage patients with COPD (correlating with hypoxemia) (28). Both nonanemic iron deficiency (26, 29) and anemia are highly prevalent in patients with COPD (8, 30, 31). Given that hepcidin production can be both modulated by the stimulatory effect of inflammation and the suppressive effect of hypoxia (27), the regulation and role of hepcidin synthesis in COPD is highly complex and requires further investigation. In addition, there are few mechanistic studies linking hepcidin, COPD, and the regulation of iron metabolism both in the lung and systemically, particularly at the molecular level.

In this study, we demonstrate that hepcidin expression in the lung is suppressed by exposure to cigarette smoke (CS) in an experimental murine model of COPD. Notably, we show that CS inhibits the amount of hepcidin produced by alveolar macrophages (AMs) in response to infection. We propose that such blunted hepcidin production by AMs limits the response of these macrophages in producing IL-6 (and TNF-α) in vivo and ex vivo, thereby limiting an appropriate immune response to respiratory infections such as Streptococcus pneumoniae. These findings may have direct translational relevance to the pathogenesis of COPD, given that the progressive course of COPD is accelerated by acute exacerbations predominantly associated with pathogens (32) such as S. pneumoniae (3335). Understanding determinants of bacterial acquisition and persistence in the lower airways in COPD is important and as yet poorly understood. Given that iron is essential for bacterial growth, respiration, and metabolism (36, 37), and that bacterial species, including S. pneumoniae, are thought to be responsible for more than 50% of exacerbation events in COPD and require iron for survival and growth (3842), our findings may provide a new investigative avenue to help identify COPD patients at high risk for infection and may provide a novel therapeutic approach to treat them.

Hamp1−/− mice were generated and provided by Dr. S. Vaulont (INSERM) (43). Hamp1−/− mice were crossed with C57BL/6J wild-type mice (strain 000664; purchased from The Jackson Laboratory) to generate Hamp1+/− mice to facilitate the production of Hamp1−/− and Hamp1+/+ littermate controls. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee at Weill Cornell Medicine, and were performed in compliance with all relevant ethical regulations.

In vivo experiments involving CS exposure used the inhalation exposure apparatus (TE-10) by Teague Enterprises with 3R4F composition cigarettes (University of Kentucky Center for Tobacco Reference Products). Age- and sex-matched mice beginning at 6–12 wk of age were exposed to CS (∼150 mg/m3) for a minimum of 3 h per day, 5 d a week, for 8 or 12 wk.

S. pneumoniae (ATCC 6303; American Type Culture Collection, Manassas, VA) was grown on 10% sheep blood agar plates (221261; BD Biosciences) overnight. An inoculating loop of bacteria was cultured in Todd Hewitt broth containing 2% yeast extract for 4–6 h (T1438; Sigma-Aldrich). Bacterial cultures were centrifuged at 15,000 × g for 1 min and resuspended in PBS. CFU were quantified by dilution of samples in Todd Hewitt broth, and titers were determined by colony counts from 1 × 103, 1 × 105, or 1 × 107 dilution. For in vivo S. pneumoniae infection, all mice were anesthetized with isoflurane (5% for induction and 2% for maintenance) prior to intranasal instillation with 1 × 106 CFU of S. pneumoniae (50 μl volume in PBS). For in vitro S. pneumoniae infection, centrifuged and resuspended bacterial cultures (multiplicity of infection = 1) were added to wells containing either isolated AMs from BAL or bone marrow–derived macrophages (BMDMs) for 2–4 h of incubation, with cells later collected for RNA extraction. Media supernatant was collected for ELISA, and CFU quantification was performed as described above.

Mice were euthanized via CO2 asphyxiation, intubated with a 20G × 1” catheter (SR-OX2025CA; Terumo), and the lungs were lavaged with ice cold PBS (10010-023; Life Technologies) supplemented with 0.5 mM EDTA (351-027-061; Quality Biological) in 0.7-ml increments for a total of 2.1 ml for cell counts, a total of 10 ml for RNA isolation, and a total of 15 ml for AM cell culture. The BAL fluid (BALF) was collected from the first 0.7 ml after centrifugation at 500 × g for 5 min at 4°C and used for ELISA and protein measurement using BCA Protein Assay Kit (23225; Thermo Fisher Scientific). The cell pellets for cell counts were treated with 1 ml of RBC Lysis Buffer (100 ml, R7757; Sigma-Aldrich) incubated for 3 min and centrifuged at 6000 × g for 3 min at 4°C. The cell pellets were then suspended in 0.5 ml of PBS and 20 μl were used for cell counting, using a hemocytometer. To assess blood count recovery, we collected peripheral blood (50 μl) in EDTA-coated capillary tubes (Thermo Fisher Scientific). Differential blood counts were measured using an automated ADVIA 120 multispecies hematology analyzer (Bayer HealthCare) calibrated for murine blood.

Mice were euthanized, BAL was performed, and necessary organs were removed and flash frozen in liquid nitrogen. BAL cells were centrifuged, resuspended in 0.35 ml TRIzol reagent (15596018; Life Technologies), and stored at −80°C. RNA isolation was performed as per the manufacturer’s instructions using TRIzol, and cleanup was performed using an RNeasy Mini Kit (74104; QIAGEN) or RNeasy Micro Kit (74004; QIAGEN). RNA concentration was measured using a NanoDrop (DS-11 Spectrophotometer; DeNovix) and converted into cDNA using the High-Capacity RNA-to-cDNA Kit (4387406; Thermo Fisher Scientific) per the manufacturer’s instructions. TaqMan Gene Expression Master Mix (4369016; Thermo Fisher Scientific) with the applicable primers for Hamp (Mm04231240_s1) or (Hs00221783_m1) for human studies; Slc40a1 (Mm01254822_m1) or (Hs00205888_m1) for human studies; Fam132b (Mm00557748_m1), Gdf15 (Mm00442228_m1), Tfrc (Mm00441941_m1), and Gapdh (Mm99999915_g1), ActinB (Mm02619580_g1), or Gnb2l1 (Hs00272002_m1) for human studies, according to the manufacturer’s instructions.

Monocyte-derived macrophages (MDM) were generated from monocytes isolated from PBMCs from smokers, subjects with COPD, and healthy nonsmokers. Subjects provided informed consent, and the study was approved by the National Research Ethics Service London–Chelsea Research Ethics Committee (study 09/H0801/85). MDMs were isolated using a Percoll gradient and adherence technique, as previously described (44). Monocytes were cultured in 2 ng/ml GM-CSF (catalog no. 7954-GM-059/CF; Bio-Techne, Abingdon, U.K.) for 12 d to generate MDMs. Macrophages were seeded at 0.5 × 106 cells per well in a 24-well plate, lysed, and RNA was extracted as described above. Lung tissue macrophages were isolated from lung parenchyma, as described previously (45). Lung tissue used in this study was assessed as being noncancerous by the pathologist and obtained from samples during tissue resection for lung cancer or emphysema.

BAL cells were collected via BAL, as previously described, for a total of 10 ml, treated with RBC lysis buffer for 3 min, and following incubation and centrifugation, resuspended in 200 μl PBS. One hundred microliters of this suspension was used for cytospin slides, which were prepared at a centrifugation of 500 × g for 5 min; the rest were used for total iron measurement. The slides were fixed using Hema 3 Stat Pack (122-911; Fisherbrand) and stained with a 4% w/v potassium ferrocyanide solution (equal volumes of potassium ferrocyanide [P3289-100G; Sigma-Aldrich]) and hydrochloric acid solution [1.2 mmol/l hydrochloric acid in distilled water]). The slides were incubated for 30 min in the working solution, rinsed in distilled water three times for 5 min, and stained with 200 μl of Nuclear Fast Red solution (N3020-100 ml; Sigma-Aldrich) for 4 min. The slides were washed one time for 5 min in distilled water and dehydrated in a series of washes with ethanol and xylene. Once dried, mounting medium (VectaMount H-5000; Vector Laboratories) was applied, a coverslip was placed, and images were taken using an EVOS FL Auto Imaging System (AMAFD1000; Life Technologies).

Commercial ELISAs were used to measure the following analytes in triplicate in homogenates or BAL of lung samples, in serum, plasma, or urine from mice, following the manufacturer’s instructions: hepcidin, (HMC-001; Intrinsic LifeSciences), erythropoietin (EPO; MEP00B; R&D Systems), IL-6 (88-7064; Invitrogen), and TNF-α (DY410-05; R&D Systems).

Tissue samples that were previously flash frozen were collected, weighed and washed with ice cold PBS, and digested with 50% nitric acid in distilled water containing 0.1% digitonin at 1/3 w/v (300410-250MG; EMD Millipore) for 2 h at 65°C, using iron-free polypropylene tubes after equilibration for 1 h at room temperature. The samples were cooled, and 30% w/w hydrogen peroxide (H1009; Sigma-Aldrich) at a 50% volume was added to the sample mixture. The samples were again digested for 1 h at 95°C and diluted with distilled water for measurement of total iron using the PinAAcle 900Z Atomic absorption spectrometer (PerkinElmer). BALF was collected as previously described, and 60 μl was used for digestion with 40 μl 50% nitric acid in distilled water containing 0.1% digitonin for 2 h at 65°C. The samples were allowed to cool, centrifuged at 6000 × g for 2 min at room temperature, and diluted with 0.2% nitric acid.

Previously frozen tissue samples were weighed and digested with nonheme iron (NHI) acid (10% trichloroacetic acid in 3 M hydrochloric acid) at a volume of 100 mg of tissue per 1 ml of acid for 20 h at 65°C. The samples were cooled to room temperature, vortexed, and centrifuged at 1000–2000 rpm for 15 s, and the supernatant was removed for analysis. Samples and iron standards (0–25 μg/ml) were added to 1-ml cuvettes containing BAT buffer (0.2% thioglycolic acid, 0.02% bathophenanthrolinedisulfonic acid disodium salt hydrate [146617; Sigma-Aldrich]) and saturated sodium acetate trihydrate (S7670; Sigma-Aldrich) in distilled water. The absorbance values were then determined using a spectrophotometer at 535 nm and compared against the standard curve.

Mice were euthanized by CO2 asphyxiation, bones were processed, and cell suspensions were filtered through a 40-μm mesh and washed in PEB (2 mM EDTA, 0.2% BSA in PBS, pH 7.4). Cell suspensions were stained with allophycocyanin-conjugated Ter119 (116212; BioLegend), allophycocyanin/Cy7–conjugated CD44 (103028; BioLegend), Hoechst (H3570; Invitrogen), and in the indicated experiments, with a modified lineage mixture, including CD3 (catalog no. 100304; BioLegend), B220 (103203; BioLegend), CD11b (101203; BioLegend), and Gr1 (108404; BioLegend) stained with streptavidin. Apoptosis in mononuclear cell suspensions was assessed using the Annexin V-FITC Apoptosis Kit (640914; BioLegend) according to manufacturer’s instructions and analyzed via flow cytometry.

Collected BALF (15 ml) was centrifuged at 500 × g for 5 min at 4°C, and the pellet was treated with RBC Lysis Buffer for 3 min. The samples were centrifuged at 6000 × g for 5 min at 4°C, resuspended in full RPMI 1640 (11875-093; Life Technologies), and added to a 12-well culture dish (one mouse per well). The cells were left to adhere overnight and treated with 2% CS extract (CSE) for 18 h, followed by 100 ng/ml LPS (tlrl-eblps; Invitrogen) for 6 h (CSE treatment of 24 h total). The primary AM media was removed (centrifuged and supernatants stored at −80°C), and the remaining cells were gently washed with PBS. Primary AMs were removed using a cell lifter with 0.35 ml of TRIzol and stored at −80°C for RNA isolation. BMDMs were extracted from the femur and tibia of mice using a 25-gauge needle attached to a 12-ml syringe filled with full DMEM in sterile conditions. The cell aggregates were broken up using an 18-gauge needle and afterward strained through a 70-μM cell strainer. The cells were centrifuged at 1000 rpm for 10 min and plated at a concentration of 1 × 106 cells per ml. The medium was changed on the third or fourth day and on the seventh was treated with LPS. CSE was prepared using unsupplemented media (either DMEM or RPMI 1640) with a VWR Chemical Transfer Pump, Variable Flow (23609170; VWR International) and reference cigarettes as previously described (46, 47). Briefly, a peristaltic pump was used to bubble mainstream smoke from five 3R4F cigarettes with filters removed through 50 ml of DMEM/RPMI. Each cigarette was smoked within 6 min until ∼17 mm remained. The extract was filter sterilized, stored at −80°C, and used immediately upon thawing. The CSE generated in this fashion was considered 100% strength and was diluted in complete DMEM/RPMI media for cell treatment. Cells pretreated with CSE (2% for 18 h, CSE treatment of 24 h total) were cultured with LPS (100 ng/ml) for 6 h or S. pneumoniae (0.5 × 106 CFU) for 2–4 h. For experiments using deferoxamine (DFO) (D9533; Sigma-Aldrich), cells were pretreated with 100 μm of DFO for 8 h. For those assessing phagocytosis, the cells were incubated at either 4 or 37°C after addition of CSE, with shorter CSE treatment times (4 and 8 h). Experiments using Chelex 100 (C7901; Sigma-Aldrich) involved treating media overnight with 10% w/v of the chelating material at 4°C, followed by removal of the supernatant for immediate use.

To characterize the functional role of the hepcidin/ferroportin axis in the pathogenesis of COPD, we used a well-established experimental model of CS-induced COPD (4850). In this model, mice exposed to CS for 6–8 wk display increased infiltration of AMs, increased markers of lung injury, and impairment of mucociliary clearance mechanisms (4850), indicative of altered inflammation and airway dysfunction. Longer exposure to CS (6 mo) results in higher mean alveolar chord length and air space diameter and increased thickness of the small airways, all established indices of experimental emphysema (47, 5052). In this study, mice exposed to 2–6 mo of CS displayed a time-dependent loss of hepcidin transcript in whole lung homogenates, with an increase in ferroportin transcript expression (Fig. 1A, 1B). Consistent with a loss of hepcidin expression and increased ferroportin expression, mice exposed to CS had higher NHI levels in whole lung homogenates, as described previously (49), suggestive of pulmonary iron overload (Fig. 1C).

FIGURE 1.

The lung ferroportin/hepcidin axis is altered in a murine model of CS-induced COPD. (A) Hamp mRNA (0 wk, n = 20; 8 wk, n = 11; 24 wk, n = 10) and (B) Slc40a1 mRNA (0 wk, n = 19; 8 wk, n = 12; and 24 wk, n = 10) in whole lung (including BAL cells) homogenates of mice exposed to room air (RA) or CS for the indicated times. (C) NHI (milligram per milliliter) in lung tissue of mice exposed to RA (0 wk) or CS (8 wk) (n = 10 per group). (D) Hepcidin expression quantified by ELISA in plasma (0 wk, n = 33; 8 wk, n = 17; 24 wk, n = 9) and (E) urine (0 wk, n = 12; 8 wk, n = 10; 24 wk, n = 4) of mice exposed to RA or CS (8 and 24 wk). (F) Hamp mRNA (0 wk, n = 22; 8 wk, n = 15; 24 wk, n = 11), (G) Slc40a1 mRNA (0 wk, n = 27; 8 wk, n = 15; 24 wk, n = 10), and (H) NHI (0 wk, n = 25; 8 wk, n = 15; 24 wk, n = 10) levels in liver tissue homogenates of mice exposed to RA or CS (8 and 24 wk). All data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005 by one-way ANOVA with Bonferroni correction. #p < 0.05 by unpaired t test. n.s., not significant.

FIGURE 1.

The lung ferroportin/hepcidin axis is altered in a murine model of CS-induced COPD. (A) Hamp mRNA (0 wk, n = 20; 8 wk, n = 11; 24 wk, n = 10) and (B) Slc40a1 mRNA (0 wk, n = 19; 8 wk, n = 12; and 24 wk, n = 10) in whole lung (including BAL cells) homogenates of mice exposed to room air (RA) or CS for the indicated times. (C) NHI (milligram per milliliter) in lung tissue of mice exposed to RA (0 wk) or CS (8 wk) (n = 10 per group). (D) Hepcidin expression quantified by ELISA in plasma (0 wk, n = 33; 8 wk, n = 17; 24 wk, n = 9) and (E) urine (0 wk, n = 12; 8 wk, n = 10; 24 wk, n = 4) of mice exposed to RA or CS (8 and 24 wk). (F) Hamp mRNA (0 wk, n = 22; 8 wk, n = 15; 24 wk, n = 11), (G) Slc40a1 mRNA (0 wk, n = 27; 8 wk, n = 15; 24 wk, n = 10), and (H) NHI (0 wk, n = 25; 8 wk, n = 15; 24 wk, n = 10) levels in liver tissue homogenates of mice exposed to RA or CS (8 and 24 wk). All data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005 by one-way ANOVA with Bonferroni correction. #p < 0.05 by unpaired t test. n.s., not significant.

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To evaluate whether changes in hepcidin and ferroportin expression in the lungs of our experimental COPD model was a ubiquitous systemic response to smoke, we assessed hepatic and systemic hepcidin regulation in response to CS exposure. Both plasma and urine levels of hepcidin decreased in response to 8 wk of CS exposure (Fig. 1D, 1E). Although plasma and urine hepcidin levels were suppressed after 6 mo of chronic CS exposure, hepatic hepcidin levels appeared to decrease at the 8-wk time point, followed by an increase in response to chronic 6-mo exposure (Fig. 1F). Hepatic ferroportin expression did not change in response to CS (Fig. 1G), despite increases in hepatic NHI levels (Fig. 1H). Typically, reduced hepcidin expression occurs as a mechanism to enhance iron absorption and recycling to increase body iron stores and availability in the plasma. Consistently, we observed that exposure to 8 wk of CS significantly increased hematocrit, hemoglobin, RBC counts, and plasma iron levels (Supplemental Fig. 1A–D).

Hepcidin production is inhibited in physiologic conditions that enhance iron requirements, such as iron deficiency anemia, tissue hypoxia (which increases iron availability for use in erythropoiesis), and/or stress erythropoiesis and dyserythropoiesis (53). To understand whether the loss of hepcidin expression in our model involved alterations in erythropoiesis, we evaluated the role of CS on erythroid development in the bone marrow. The cell surface markers CD71/CD44 and Ter119 were used to characterize erythroblast populations in freshly isolated bone marrow from smoke- and room air–exposed mice. After 8 wk of smoke, the number of Ter119+ cells increased (although NS) in the marrow, with increases across all erythroid progenitors and particularly significant increases in the EIV/EV.44 populations, suggestive of expanded erythropoiesis (Fig. 2A, 2B).

FIGURE 2.

Mice exposed to CS have expanded but iron-deficient erythropoietic activity. (A) Flow cytometry gating strategy using lineage-negative erythroid (LinE) (CD3, B220, Gr1, CD11b), CD44, Ter119, and Hoechst (DNA) to characterize erythroid maturation with quantification of (B) Ter119+ cells (left panel) and number of cells per erythropoietic development stage (right panel) in the bone marrow of mice exposed to room air (RA) or CS for 8 wk. (C) Schematic of erythropoiesis and EFRE regulation on hepcidin. (D) EPO levels quantified by ELISA in plasma (0 wk, n = 6; 8 wk, n = 13) and (E) Fam132b mRNA expression (n = 8 per group) in bone marrow of wild-type mice exposed to RA or CS (8 wk). (F and G) Representative flow cytometry data showing annexin V staining in (F) marrow and (G) Ter119+ populations isolated from RA- and CS-exposed mice (8 wk). (H) Total iron levels in the bone marrow with RBCs (left panel) and without RBCs (right panel) (n = 8 per group). (I) Slc40a1 mRNA (left panel) (n = 7 per group) and Tfrc mRNA (right panel) (0 wk, n = 14; 8 wk n = 13) levels in the bone marrow of mice exposed to either RA or CS (8 wk). All data are mean ± SEM. **p < 0.01 by one-way ANOVA with Tukey post hoc test. ##p < 0.01, ###p < 0.001 by Student unpaired t test. FMO, fluorescence minus one; n.s., not significant.

FIGURE 2.

Mice exposed to CS have expanded but iron-deficient erythropoietic activity. (A) Flow cytometry gating strategy using lineage-negative erythroid (LinE) (CD3, B220, Gr1, CD11b), CD44, Ter119, and Hoechst (DNA) to characterize erythroid maturation with quantification of (B) Ter119+ cells (left panel) and number of cells per erythropoietic development stage (right panel) in the bone marrow of mice exposed to room air (RA) or CS for 8 wk. (C) Schematic of erythropoiesis and EFRE regulation on hepcidin. (D) EPO levels quantified by ELISA in plasma (0 wk, n = 6; 8 wk, n = 13) and (E) Fam132b mRNA expression (n = 8 per group) in bone marrow of wild-type mice exposed to RA or CS (8 wk). (F and G) Representative flow cytometry data showing annexin V staining in (F) marrow and (G) Ter119+ populations isolated from RA- and CS-exposed mice (8 wk). (H) Total iron levels in the bone marrow with RBCs (left panel) and without RBCs (right panel) (n = 8 per group). (I) Slc40a1 mRNA (left panel) (n = 7 per group) and Tfrc mRNA (right panel) (0 wk, n = 14; 8 wk n = 13) levels in the bone marrow of mice exposed to either RA or CS (8 wk). All data are mean ± SEM. **p < 0.01 by one-way ANOVA with Tukey post hoc test. ##p < 0.01, ###p < 0.001 by Student unpaired t test. FMO, fluorescence minus one; n.s., not significant.

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Increases in erythropoietic activity suppress hepcidin, at least in part via the erythroblast-secreted factors erythroferrone (EFRE; encoded by the gene FAM132b) (53), growth differentiation factor 15 (GDF15) (54), or twisted gastrulation (TWSG1) (55) (Fig. 2C). EFRE, GDF15, and TWSG1 rapidly suppress hepcidin to allow iron acquisition from absorption and storage sites, thus favoring recovery from anemia secondary to blood loss and inflammation (56). We therefore evaluated the role of CS on the production of EPO, EFRE, GDF15, and TWSG1 in our murine COPD model. EPO levels increased in both the plasma and kidneys of mice exposed to CS (8 wk), whereas hepatic EPO levels did not change in response to CS (Fig. 2D, Supplemental Fig. 1E, 1F). EFRE (Fam132b) expression significantly increased in murine bone marrow exposed to CS, whereas GDF15 levels decreased (Fig. 2E, Supplemental Fig. 1G). We did not observe any change in TWSG1 expression levels upon CS exposure (Supplemental Fig. 1H). These data suggest that hepcidin may be chronically suppressed by the exuberant but ineffective erythropoietic activity (ineffective erythropoiesis) induced by CS. Consistently, we observed increased expression of annexin V+ cells in the whole bone marrow fraction, as well as in the Ter119+ population (Fig. 2F, 2G), suggesting increased activation of apoptosis. To investigate whether such changes in erythropoiesis were related to changes in iron handling by erythrocytes, we measured the amount of iron in the bone marrow (with or without RBCs) of mice exposed to CS. Although CS exposure did not change the iron content of the total bone marrow, including RBCs, CS significantly reduced iron levels in the non-RBC population as well as reducing the expression of ferroportin (Fig. 2H, 2I). These data suggest that there may be a high iron requirement by the bone marrow that is not being fulfilled (iron-deficient erythropoiesis), confirmed by increases in transferrin receptor gene expression (Fig. 2I).

The mononuclear phagocyte system (MPS), which encompasses monocytes and macrophages, is a multifunctional system beyond immunity and tissue repair that also serves to control the body’s metabolic needs for iron. In conditions of expanded but inefficient erythropoiesis, such as that observed in our murine smoke model, iron-recycling macrophages may be mobilized to recycle senescent RBCs, detoxify free heme, and acquire iron for de novo hemoglobin synthesis (57). AMs are the most abundant APCs in the lung, regulating immune responses, including phagocytosis of particulate matter, secretion of cytokines and enzymes, and control of microbes along with other homeostatic functions (17). During infection, inflammatory signals stimulate the production of hepcidin, resulting in the loss of ferroportin and reduction of extracellular iron, which is thought to be a general defense mechanism against invading pathogens (58, 59). AMs from COPD patients and smokers are dysfunctional, with altered inflammatory responses (6063) and lower phagocytic ability (64). This may have severe implications for AM function in the lung and in the pathogenesis of COPD, in which recurrent bacterial infections are ubiquitous (65). Previous studies have demonstrated increased prevalence of iron-loaded AMs in smokers and subjects with COPD; however, the biological relevance of such observations remains to be elucidated (66, 67). In this study, we first assessed whether hepcidin and ferroportin are altered in lung tissue-derived and MDM of nonsmokers, smokers, and subjects with COPD. Smokers had higher hepcidin expression in tissue-derived macrophages but not in MDMs when compared with healthy controls (Fig. 3A, 3C). Individuals with COPD trended toward higher hepcidin expression in tissue-derived macrophages and in MDMs when compared with healthy controls; however, these changes were not statistically significant (Fig. 3A, 3C). Smokers also had higher ferroportin expression in tissue-derived and MDMs when compared with healthy controls (Fig. 3B, 3D). Individuals with COPD had lower ferroportin expression in both tissue-derived and MDMs when compared with smokers; however, these changes were not statistically significant (Fig. 3B, 3D). The above results demonstrate that hepcidin and ferroportin are increased by smoke, an effect that may be sustained in tissue resident macrophages and MDMs from individuals with COPD.

FIGURE 3.

The ferroportin/hepcidin macrophage axis is altered in smokers and in subjects with COPD. (A) HAMP mRNA (nonsmoker, n = 5; smoker, n = 6; COPD n = 9), (B) SLC40A1 mRNA (nonsmoker, n = 5; smoker, n = 6; COPD, n = 8) expression levels in lung-derived macrophages from lung tissue macrophages isolated from lung parenchyma. (C) HAMP mRNA (nonsmoker, n = 7; smoker, n = 5; COPD n = 8) and (D) SLC40A1 mRNA (nonsmoker, n = 7; smoker, n = 6; COPD n = 9) expression levels of MDM generated from monocytes isolated from PBMCs stimulated with GM-CSF (2 ng/ml GM-CSF for 12 d). All data are mean ± SEM. *p < 0.05 by Kruskal–Wallis test, followed by Dunn post hoc test.

FIGURE 3.

The ferroportin/hepcidin macrophage axis is altered in smokers and in subjects with COPD. (A) HAMP mRNA (nonsmoker, n = 5; smoker, n = 6; COPD n = 9), (B) SLC40A1 mRNA (nonsmoker, n = 5; smoker, n = 6; COPD, n = 8) expression levels in lung-derived macrophages from lung tissue macrophages isolated from lung parenchyma. (C) HAMP mRNA (nonsmoker, n = 7; smoker, n = 5; COPD n = 8) and (D) SLC40A1 mRNA (nonsmoker, n = 7; smoker, n = 6; COPD n = 9) expression levels of MDM generated from monocytes isolated from PBMCs stimulated with GM-CSF (2 ng/ml GM-CSF for 12 d). All data are mean ± SEM. *p < 0.05 by Kruskal–Wallis test, followed by Dunn post hoc test.

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We next wished to assess whether smoke altered the production of hepcidin in response to infection in AMs. First, we assessed hepcidin expression in AMs isolated from the BALF of animals that were exposed to smoke for 8 and 12 wk. CS exposure alone did not considerably alter hepcidin expression in BAL AMs. Similarly, CSE, an in vitro model of CS exposure) did not considerably alter hepcidin expression in primary AMs or BMDMs (Fig. 4A, Supplemental Fig. 2A–C). However, primary AMs exposed to CSE prior to the addition of the Gram-negative molecule LPS showed blunted Hamp gene expression and blunted Il-6 gene expression and release (Fig. 4A, 4B); BMDMs also had a blunted hepcidin response to LPS with CSE (Supplemental Fig. 2C). CS exposure significantly increased ferroportin expression in BAL AMs and in primary AMs or BMDMs treated with CSE (Supplemental Fig. 2A–C). LPS treatment reduced ferroportin expression, confirming that AMs may try to sequester iron in response to bacterial infection. Treatment of CSE prior to LPS appeared to inhibit the loss of ferroportin in BMDMs but seemed to have little effect in AMs (Fig. 4, Supplemental Fig. 2B, 2C). Notably, the doses and exposure times for CSE used in this study did not alter the viability of AMs or BMDMs (Supplemental Fig. 2D). These data suggest that macrophages exposed to CS may no longer be able to sequester iron from bacterial pathogens, which may lead to increased susceptibility to infection.

FIGURE 4.

The ferroportin/hepcidin macrophage axis is altered in a murine model of CS-induced COPD. (A) Hamp mRNA, (B) il-6 mRNA (top panel, n = 3 per group), and IL-6 in media supernatant (bottom panel) quantified by ELISA; (C) Slc40a1 mRNA in primary AMs isolated from wild-type mice, adherence purified, and treated with 2% CSE for 18 h (CSE treatment of 24 h total) followed by 100 ng/ml LPS (6 h). Data are representative of three to four independent experiments. All data are mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.001 by one-way ANOVA with Bonferroni correction. ##p < 0.1 by Student unpaired t test. n.s., not significant.

FIGURE 4.

The ferroportin/hepcidin macrophage axis is altered in a murine model of CS-induced COPD. (A) Hamp mRNA, (B) il-6 mRNA (top panel, n = 3 per group), and IL-6 in media supernatant (bottom panel) quantified by ELISA; (C) Slc40a1 mRNA in primary AMs isolated from wild-type mice, adherence purified, and treated with 2% CSE for 18 h (CSE treatment of 24 h total) followed by 100 ng/ml LPS (6 h). Data are representative of three to four independent experiments. All data are mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.001 by one-way ANOVA with Bonferroni correction. ##p < 0.1 by Student unpaired t test. n.s., not significant.

Close modal

Iron uptake by macrophages may occur via phagocytosis of senescent RBCs or via receptor-mediated endocytosis mechanisms involving receptors such as transferrin receptor, divalent metal transporter 1 (DMT1), or zinc transporter ZIP14 (SLC39A14). Both phagocytosis and receptor-mediated endocytosis can be nonspecifically inhibited by reducing the temperature of cultures to 4°C (68, 69). To assess whether phagocytosis or endocytosis alters the expression of hepcidin and ferroportin by smoke, we cultured AMs and BMDMs treated with CSE at 4°C and found that these conditions inhibit the loss of hepcidin and increase ferroportin expression induced by smoke (with or without LPS) when compared with BMDMs grown at 37°C (Supplemental Fig. 2B, 2C). Removal of iron from CSE using Chelex 100 (a styrene–divinylbenzene copolymer containing iminodiacetic acid groups that bind to transition metal ions, including iron) (Supplemental Fig. 2E) had no effect on the expression of hepcidin or ferroportin induced by smoke (Supplemental Fig. 2F). Incubating cells with the iron chelator DFO trended toward inhibiting the effects of CSE on LPS-induced hepcidin expression and LPS-induced loss of ferroportin expression (Supplemental Fig. 2G). These results suggest that CS alters the innate response of macrophages to produce hepcidin and lower ferroportin in response to infection, a phenomenon that may involve the regulation of iron via phagocytosis or endocytosis.

To investigate the physiological function of hepcidin in the lung and the physiological response of the lung to smoke-induced injury and inflammation, we used a murine model of hepcidin deficiency. As expected, Hamp−/− mice had lower levels of lung and AM Hamp expression and higher levels of lung and AM Slc40A1 expression (Fig. 5A, 5B, Supplemental Fig. 2G, 2H). Hamp−/− mice also had higher NHI and higher AM total iron levels as determined by atomic absorption spectroscopy as well as Perls’ staining (Fig. 5C–E). Using a murine model of experimental COPD, exposing Hamp−/− mice to CS for 8 wk resulted in similar numbers of total BALF cell infiltrates; however, there was a significantly lower number of infiltrating macrophages in the Hamp−/− mice in response to smoke, despite the Hamp−/− mice having higher baseline numbers of macrophages (Fig. 5F, 5G). Interestingly, at baseline, Hamp−/− mice had significantly more BAL protein, suggestive of increased injury (Fig. 5H). In response to smoke, Hamp−/− mice appeared to have more BALF protein; however, this was NS when compared with Hamp+/+ mice exposed to CS (Fig. 5H). Although iron levels were higher at baseline in the Hamp−/− mice, there was no difference between Hamp+/+ and Hamp−/− mice lung or AM iron content upon smoke exposure (Fig. 5C, 5D). Similarly, ferroportin levels were higher at baseline in the Hamp−/− mice, but there was no difference between ferroportin expression in the Hamp+/+ and Hamp−/− mice upon smoke exposure (Fig. 5B). Collectively, the above data imply that loss of hepcidin in the lung alters baseline iron and ferroportin levels, but this is not exacerbated by smoke.

FIGURE 5.

Loss of hepcidin in vivo does not alter smoke-induced injury, iron changes, or ferroportin expression. (A) Hamp mRNA expression in BAL AMs of Hamp+/+ (n = 4) and Hamp−/− (n = 7) mice. (B) Slc40a1 mRNA expression in BAL AMs. (C) NHI levels (microgram per milliliter per milligram) in whole lung tissue (excluding BAL cells) of Hamp+/+ (room air [RA], n = 8; CS, n = 9) and Hamp−/− (RA, n = 5; CS n = 8) and (D) total iron levels in BAL AMs of Hamp+/+ (RA and CS, n = 5) and Hamp−/− (RA, n = 3; CS, n = 5) mice exposed to RA or CS (8 wk). (E) Representative Perls’ staining of BAL AMs of Hamp+/+ and Hamp−/− mice exposed to RA or CS (8 wk) (arrows indicate presence of ferric iron in AMs) Scale bar, 50 μm. (F) Total infiltrating leukocyte cell counts of Hamp+/+ (RA and CS, n = 12) and Hamp−/− (RA, n = 9; CS, n = 12) and (G) total macrophage counts calculated from H&E cytospins of BAL cells of Hamp+/+ (n = 3 per group) and Hamp−/− (n = 2 per group) mice exposed to RA and CS (8 wk). (H) BALF protein (milligram) in Hamp+/+ (RA, n = 11; CS, n = 13) and Hamp−/− (RA, n = 8; CS, n = 12) of mice exposed to RA or CS (8 wk). All data are mean ± SEM. **p < 0.01, ***p < 0.005, ****p < 0.001 by one-way ANOVA followed by Tukey posthoc test. #p < 0.05, ####p < 0.001 by Student unpaired t test. n.s., not significant.

FIGURE 5.

Loss of hepcidin in vivo does not alter smoke-induced injury, iron changes, or ferroportin expression. (A) Hamp mRNA expression in BAL AMs of Hamp+/+ (n = 4) and Hamp−/− (n = 7) mice. (B) Slc40a1 mRNA expression in BAL AMs. (C) NHI levels (microgram per milliliter per milligram) in whole lung tissue (excluding BAL cells) of Hamp+/+ (room air [RA], n = 8; CS, n = 9) and Hamp−/− (RA, n = 5; CS n = 8) and (D) total iron levels in BAL AMs of Hamp+/+ (RA and CS, n = 5) and Hamp−/− (RA, n = 3; CS, n = 5) mice exposed to RA or CS (8 wk). (E) Representative Perls’ staining of BAL AMs of Hamp+/+ and Hamp−/− mice exposed to RA or CS (8 wk) (arrows indicate presence of ferric iron in AMs) Scale bar, 50 μm. (F) Total infiltrating leukocyte cell counts of Hamp+/+ (RA and CS, n = 12) and Hamp−/− (RA, n = 9; CS, n = 12) and (G) total macrophage counts calculated from H&E cytospins of BAL cells of Hamp+/+ (n = 3 per group) and Hamp−/− (n = 2 per group) mice exposed to RA and CS (8 wk). (H) BALF protein (milligram) in Hamp+/+ (RA, n = 11; CS, n = 13) and Hamp−/− (RA, n = 8; CS, n = 12) of mice exposed to RA or CS (8 wk). All data are mean ± SEM. **p < 0.01, ***p < 0.005, ****p < 0.001 by one-way ANOVA followed by Tukey posthoc test. #p < 0.05, ####p < 0.001 by Student unpaired t test. n.s., not significant.

Close modal

The above results suggest that loss of hepcidin may not alter the injurious response of the lung to CS but may alter the number of BALF macrophages infiltrating the lung. To address whether loss of hepcidin alters the functional immune response of the lung to smoke, we assessed IL-6 production. Loss of hepcidin resulted in less BALF IL-6 at baseline and in response to smoke (Fig. 6A). To assess whether hepcidin regulated the functional response of AMs with or without smoke, we isolated primary AMs from Hamp−/− and Hamp+/+ mice and treated them with CSE, followed by LPS stimulation. IL-6 was robustly induced by LPS and decreased by CSE in primary Hamp+/+ AMs. AMs isolated from Hamp−/− mice had impaired LPS-induced IL-6 production (Fig. 6B, 6C). Similarly, loss of hepcidin in AMs exacerbated CS-induced inhibition of LPS-associated IL-6, suggesting that hepcidin may play a role in the mechanism by which CS inhibits IL-6 responses by LPS and by CS (Fig. 6B, 6C).

FIGURE 6.

AMs and mice deficient in Hamp have altered immune responses to S. pneumoniae infection. (A) Fold change in IL-6 levels in the BALF of Hamp+/+ (room air [RA], n = 8; CS, n = 9) and Hamp−/− (RA, n = 5; CS, n = 8) mice exposed to RA and CS (8 wk) measured by ELISA. (B) Il-6 mRNA and (C) IL-6 levels (picograms per milliliter by ELISA) in the media of primary AMs isolated from Hamp+/+ and Hamp−/− mice treated with 2% CSE for 18 h (CSE treatment of 24 h total) followed by 100 ng/ml LPS (6 h). Data representative of three to four independent experiments. (D) Bacterial titer (left panel) at 1 × 103 and 1 × 105 dilutions with the number of CFUs quantified (right panel) in the media supernatants of Hamp+/+ and Hamp−/− primary AMs treated with 0.5 × 106 CFU S. pneumoniae for 2 or 4 h. Data are representative of n = 4–6 independent experiments. (E) Il-6 mRNA expression, (F) IL-6 levels (picograms per milliliter by ELISA), and (G) TNF-α levels (picograms per milliliter by ELISA) in primary AMs and corresponding media supernatants from Hamp+/+ and Hamp−/− mice treated with 2% CSE (24 h) followed by S. pneumoniae (4 h). Data are representative of n = 2 independent experiments. (H and I) Relative weights, (J) percentage of macrophages in BALF by H&E staining, and (K) BALF IL-6 levels (picograms per milliliter by ELISA) in Hamp+/+ and Hamp−/− (n = 5 per group) mice at 0, 18, 24, and 48 h postinfection of 1 × 105 and 1 × 109 CFU of S. pneumoniae after exposure to either (H) RA or (I) CS (8 wk). All data are mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.001 by one-way ANOVA followed by Tukey posthoc test. #p < 0.05 by Student unpaired t test.

FIGURE 6.

AMs and mice deficient in Hamp have altered immune responses to S. pneumoniae infection. (A) Fold change in IL-6 levels in the BALF of Hamp+/+ (room air [RA], n = 8; CS, n = 9) and Hamp−/− (RA, n = 5; CS, n = 8) mice exposed to RA and CS (8 wk) measured by ELISA. (B) Il-6 mRNA and (C) IL-6 levels (picograms per milliliter by ELISA) in the media of primary AMs isolated from Hamp+/+ and Hamp−/− mice treated with 2% CSE for 18 h (CSE treatment of 24 h total) followed by 100 ng/ml LPS (6 h). Data representative of three to four independent experiments. (D) Bacterial titer (left panel) at 1 × 103 and 1 × 105 dilutions with the number of CFUs quantified (right panel) in the media supernatants of Hamp+/+ and Hamp−/− primary AMs treated with 0.5 × 106 CFU S. pneumoniae for 2 or 4 h. Data are representative of n = 4–6 independent experiments. (E) Il-6 mRNA expression, (F) IL-6 levels (picograms per milliliter by ELISA), and (G) TNF-α levels (picograms per milliliter by ELISA) in primary AMs and corresponding media supernatants from Hamp+/+ and Hamp−/− mice treated with 2% CSE (24 h) followed by S. pneumoniae (4 h). Data are representative of n = 2 independent experiments. (H and I) Relative weights, (J) percentage of macrophages in BALF by H&E staining, and (K) BALF IL-6 levels (picograms per milliliter by ELISA) in Hamp+/+ and Hamp−/− (n = 5 per group) mice at 0, 18, 24, and 48 h postinfection of 1 × 105 and 1 × 109 CFU of S. pneumoniae after exposure to either (H) RA or (I) CS (8 wk). All data are mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.001 by one-way ANOVA followed by Tukey posthoc test. #p < 0.05 by Student unpaired t test.

Close modal

One of the most common organisms cultured from sputum and bronchoscopic samples of patients with COPD is S. pneumoniae (35). Both smokers and individuals with COPD have an increased susceptibility to S. pneumoniae infection (33, 34), and bacterial colonization and infection by S. pneumoniae typically occurs early in the course of COPD (35). To assess whether loss of hepcidin alters the response of AMs to S. pneumoniae, primary AMs and BMDMs isolated from Hamp+/+ and Hamp−/− mice were cultured and treated with live S. pneumoniae (strain 6303; American Type Culture Collection). AMs deficient in Hamp were less able to clear S. pneumoniae with a greater burden of CFU remaining in the supernatants 2–4 h postinfection (Fig. 6D). Similarly, AMs isolated from Hamp−/− mice had impaired S. pneumoniae–induced IL-6 and TNF-α production at baseline and in the presence of CSE (Fig. 6E–G). In vivo, Hamp−/− mice infected with S. pneumoniae displayed signs of a higher bacterial burden reflected by more significant loss in weight, higher numbers of BALF macrophage infiltrates, and lower AM Il-6 levels (Fig. 6H–K, Supplemental Fig. 2I, 2J). Hamp−/− mice exposed to smoke followed by S. pneumoniae infection also had more severe loss in body weight and heightened BALF macrophage infiltrates (Fig. 6I, 6J).

Changes in pulmonary and systemic iron regulation are associated with the progression and pathogenesis of COPD. In this study, we show that the master iron hormone hepcidin is lower in whole lung homogenates, plasma, and urine of animals exposed to smoke. We show that a loss in hepcidin expression may be associated with increased levels of EPO, EFRE, and expanded but inefficient erythropoiesis in the bone marrow. We hypothesize that this may in turn lead to changes in iron uptake and release in macrophages of the lung. Consistently, murine AMs exposed to smoke have higher ferroportin and higher iron levels. Upon exposure to smoke, AMs fail to increase hepcidin in response to Gram-negative or Gram-positive infection, and loss of hepcidin in vivo results in blunted functional responses of AMs and altered responses to S. pneumoniae infection.

These results support a theory whereby a rise in hepcidin expression is an essential bacteriostatic response of AMs, consistent with previous reports that inflammatory signals stimulate the production of hepcidin (58). Our observed increases in hepcidin in macrophages derived from healthy smokers and subjects with COPD is also consistent with hepcidin activation in patients with mild-to-moderate disease or during an exacerbation event [commonly associated with infection (70, 71)], reflective of the proinflammatory phenotype of AMs in smokers and individuals with COPD (26, 27). Similarly, our observations that whole lung and systemic hepcidin levels decline with chronic smoking is consistent with the loss of hepcidin expression in severe end-stage COPD patients, most likely correlating with hypoxemia (28). These findings further highlight that hepcidin production can be both modulated by the stimulatory effect of inflammation and the suppressive effect of hypoxia (27).

Although a rise in hepcidin in macrophages appears to be a protective response to infection, whether this rise in hepcidin is directly related to the effect of hepcidin on ferroportin expression, and in turn iron export, requires further extensive investigation. Notably, we were unable to validate the transcript levels of ferroportin on AMs observed in this study by immunoblotting because of our inability to validate commercially available murine ferroportin Abs for immunoblotting (data not shown). Preliminary data generated by this study suggest that the effect of smoking on ferroportin expression and/or iron changes may be independent of hepcidin expression. In addition, loss of hepcidin expression in the lung does not alter the injurious response of the lung to smoke. Irrespective of this, cigarette smoking impairs innate hepcidin induction in macrophages, which reduces their ability to respond to infection. AMs are plastic cells with many important roles in the lung, including clearance of inhaled particulates and microbes and modulation of inflammation and tissue repair (72, 73). Defects in the ability of these cells to adapt their intracellular metabolism may alter their ability to produce inflammatory cytokines and reactive oxygen species and to perform other defense and repair functions (74). The observed impairments in AM responses may lead to an inefficient adaptive response to environmental cues, such as the presence of bacteria, resulting in an increased microbial burden in COPD, enhancing the risk of recurrent infections. Bacterial colonization and impaired clearance are characteristic of COPD and are associated with disease progression (65). Increased rates of recurrent infection in COPD may manifest as chronic bronchitis and/or excessive mucus production and are associated with reduced quality of life, more rapid decline in lung function, and increased mortality (70, 71).

Our results also highlight the important systemic effects cigarette smoking has on erythropoiesis and iron regulation. We demonstrate that exposure to smoke results in expanded but inefficient erythropoietic activity, most likely due to the decline in iron levels in erythropoietic precursors. Although there is some evidence to suggest that patients with COPD have altered responses to hypoxia and may have higher EPO levels (75) and that chronic CS increases the size of the mitotic and postmitotic pools in the marrow (76), little is known about the direct effects of CS on erythropoietic function or EPO production. These findings suggest that under acute hypoxic conditions dietary iron absorption by enterocytes and release by macrophages cannot match the increased requirements for erythropoiesis. Defects in iron reuse, ineffective erythropoiesis, and impaired marrow response to normal EPO levels have all been incriminated in the pathogenesis of anemia, of which there is an increased prevalence in subjects with severe COPD (8, 30, 31). Further studies investigating the effects of CS on the production of hematopoietic precursors supplying the MPS and the role of iron in this process will be of great interest for understanding macrophage defects in subjects with COPD. In addition, more extensive studies using pharmacological inhibitors of phagocytosis and endocytosis are required to fully understand the role that iron-associated phagocytosis has in this process.

This work provides an early foundation for further studies to understand the role of hepcidin in lung biology and disease as well as the interplay between the bone marrow, erythropoiesis, and the MPS in the lung. Mechanistic studies assessing the ferroportin-independent role of hepcidin in the lung in response to infection and various environmental conditions are required and are the subject of further investigation. Our findings suggest that a loss of hepcidin systemically and at the level of the AM (in response to infection) may play an important role in the pathogenesis and progression of COPD. In particular, given that iron is essential to bacterial growth, respiration, and metabolism (36, 37) and that the bacterial species thought to be responsible for more than 50% of exacerbation events in COPD, including S. pneumoniae, require iron for survival and growth (3842), understanding the regulation of iron metabolism in the lung microenvironment and interaction with the immune system is of great importance. To conclude, if replicated, our findings that hepcidin repression in the lung upon smoke exposure may alter the bactericidal response of the lung macrophages provides a new investigative avenue to help identify, understand, and treat COPD patients at high risk for infection.

We thank Dr. Gregory Sonnenberg, Dr. William Zhang, and Dr. Augustine M.K. Choi for critical insight and discussion.

This work was supported by National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute Grant R00-HL125899 (to S.M.C.), NIH/National Institute on Aging Grants R01AG052530 (to H.S.-D.) and R01AG056699 (to H.S.-D.), Science Foundation Ireland Grant FRL4862 (to S.M.C.), and a European Respiratory Society short-term research fellowship (to J.R.B.). This work was also supported by the National Institute for Health Research, Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield National Health Service Foundation Trust, and Imperial College London.

The online version of this article contains supplemental material.

Abbreviations used in this article:

AM

alveolar macrophage

BAL

bronchoalveolar lavage

BALF

BAL fluid

COPD

chronic obstructive pulmonary disease

CS

cigarette smoke

CSE

CS extract

DFO

deferoxamine

EFRE

erythroferrone

EPO

erythropoietin

GDF15

growth differentiation factor 15

MDM

monocyte-derived macrophage

MPS

mononuclear phagocyte system

NHI

nonheme iron

TWSG1

twisted gastrulation.

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The authors have no financial conflicts of interest.

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