Macrophage Rac2 Is Required to Reduce the Severity of Cigarette Smoke-induced Pneumonia

Abstract

Rationale: Cigarette smoking is prevalent in the United States and is the leading cause of preventable diseases. A prominent complication of smoking is an increase in lower respiratory tract infections (LRTIs). Although LRTIs are known to be increased in subjects that smoke, the mechanism(s) by which this occurs is poorly understood.

Objectives: Determine how cigarette smoke (CS) reduces reactive oxygen species (ROS) production by the phagocytic NOX2 (NADPH oxidase 2), which is essential for innate immunity in lung macrophages.

Methods: NOX2-derived ROS and Rac2 (Ras-related C3 botulinum toxin substrate 2) activity were determined in BAL cells from wild-type and Rac2^-/- mice exposed to CS or cadmium and in BAL cells from subjects that smoke. Host defense to respiratory pathogens was analyzed in mice infected with Streptococcus pneumoniae.

Measurements and main results: NOX2-derived ROS in BAL cells was reduced in mice exposed to CS via inhibition of the small GTPase Rac2. These mice had greater bacterial burden and increased mortality compared with air-exposed mice. BAL fluid from CS-exposed mice had increased levels of cadmium, which mediated the effect on Rac2. Similar observations were seen in human subjects that smoke. To support the importance of Rac2 in the macrophage immune response, overexpression of constitutively active Rac2 by lentiviral administration increased NOX2-derived ROS, decreased bacterial burden in lung tissue, and increased survival compared with CS-exposed control mice.

Conclusions: These observations suggest that therapies to maintain Rac2 activity in lung macrophages restore host defense against respiratory pathogens and diminish the prevalence of LRTIs in subjects that smoke.

Keywords: NADPH oxidase 2; heavy metals; innate immunity; respiratory tract infections.

Figure 1.

Cigarette smoke (CS) increases lung bacterial burden. (A) Total number of BAL cells (n = 6), (B) cell differential (n = 6), and (C–E) percentage of resident macrophages (CD11b^lo/F4/80⁺/CD11c^hi) (n = 3) (C), recruited macrophages (CD11b⁺/F4/80⁺/CD11c⁻/CD206⁻/CCR2⁺/Ly6C⁺) (n = 3) (D), and early inflammatory monocytes (CCR2⁺/Ly6C^hi) (n = 3) (E) from air- and CS-exposed mice (16 cigarettes/d for 10 d). (F) Lung macrophage NOX2 (NADPH oxidase 2)-derived reactive oxygen species (ROS) generation (n = 6) and (G) lung colony-forming units (cfu) from air- and CS-exposed mice infected with vehicle or Streptococcus pneumoniae (10⁷ cfu for 48 h, strain A66.1, type 3) (n = 5). (H) Cell differential, total number (n = 7–8), (I) lung macrophage NOX2-derived ROS generation (n = 5), and (J) lung colony-forming units from mice treated with control or clodronate liposomes and infected with vehicle or S. pneumoniae (n = 7–8). *P < 0.05; **P < 0.001; ***P < 0.0001. Values shown represent means ± SEM. Two-tailed t test statistical analysis was used for A, G, and J. Mann-Whitney U statistical analysis was used for C–E. One-way ANOVA followed by Tukey’s multiple comparison test was performed on B, F, H, and I. Results from A, B, F, and G were repeated at least five times; C–E were conducted once with representative plots of three shown, and H–J were repeated three times. BALC = BAL cells; Clod = clodronate; FITC = fluorescein isothiocyanate; Lymph = lymphocyte; Mac = macrophage/monocyte; PMN = polymorphonuclear neutrophil; S.p. = Streptococcus pneumoniae.

Figure 2.

Macrophage Rac2 (Ras-related C3 botulinum toxin substrate 2) regulates NOX2 (NADPH oxidase 2)-derived reactive oxygen species (ROS). (A and B) Membrane-derived ROS generation in THP-1 cells treated with the indicated concentration of cigarette smoke extract (CSE) for 3 hours (n = 3) (A) and THP-1 cells expressing scrambled or gp91^phox siRNA treated with vehicle or CSE (5%) for 3 hours (n = 3) (B). (B, inset) Immunoblot analysis confirming gp91^phox silencing. (C and D) Immunoblot analysis of NOX2 complex proteins in isolated membrane or cytosol fractions from MH-S cells treated with the indicated concentrations of CSE (C) and THP-1 cells expressing scrambled or Rac2 siRNA treated with vehicle or CSE (D). (E) Immunoblot analysis of THP-1 cells expressing scrambled or p67^phox siRNA treated with vehicle or CSE. (F) Histidine (His) pull-down in MH-S cells expressing empty or p67^phox-V5-His and treated with vehicle or CSE and (G) quantification. (H) Immunoblot analysis of BAL cells from air- or CS-exposed mice (n = 3). (I and J) Rac2 activity in BAL cells from air- and CS-exposed mice infected with vehicle or Streptococcus pneumoniae (n = 4) (I) and mice treated with control or clodronate liposomes and infected with vehicle or S. pneumoniae (n = 4) (J). (K) Rac2 membrane activity in BAL cells from control and anti-Ly6G–treated mice infected with vehicle or S. pneumoniae (n = 5–6). **P < 0.001; ***P < 0.0001. Values shown represent means ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test was used for A, B, and I–K. Results from A, B, and J were repeated three times; immunoblot analyses were performed three times, with representative immunoblots shown in C–F and H; I was conducted at least five times. Cyto = cytosol; IB = immunoblot; Mem = membrane; PD = pull-down; Scr = scrambled; S.p. = Streptococcus pneumoniae.

Figure 3.

Rac2^−/− mice have impaired host defense. Wild-type and Rac2^−/− mice were infected with Streptococcus pneumoniae. (A) Rac2 activity in BAL cell membrane (n = 4–5), (B) immunoblot analysis in cytosol and membrane fractions of BAL cells, and (C) NOX2 (NADPH oxidase 2)-derived reactive oxygen species generation in BAL cell membrane (n = 5). (D) Hematoxylin and eosin staining of lung tissues was performed (n = 4–5); and (E) lung colony-forming units (n = 9) and (F) Kaplan-Meier survival curves (n = 8–9) were determined. Scale bars, 600 μm. The second and fourth rows in D show magnifications of the boxed regions in the first and third rows, respectively. **P < 0.001; ***P < 0.0001. Values shown represent means ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test was used for A and C; two-tailed Student’s t-test statistical analysis was used for E, and a log-rank (Mantel-Cox) test was used for F. Results from A–F were repeated three times with representative immunoblot and histology micrographs shown. C = cytosol; M = membrane; Rac2 = Ras-related C3 botulinum toxin substrate 2; S.p. = Streptococcus pneumoniae; WT = wild type.

Figure 4.

Cadmium in cigarette smoke (CS) impairs BAL cell innate immunity via Rac2 inhibition. (A) BAL fluid samples from air- and CS-exposed wild-type mice were subjected to inductively coupled plasma mass spectrometry to determine concentrations of the indicated metals (n = 4). Wild-type mice were treated with vehicle or CdCl₂ (100 ng/kg intratracheally for 5 d) and then infected with Streptococcus pneumoniae. (B) Rac2 (Ras-related C3 botulinum toxin substrate 2) activity in BAL cell membrane (n = 3–4), (C) immunoblot analysis in cytosol and membrane fractions of BAL cells, and (D) NOX2 (NADPH oxidase 2)-derived reactive oxygen species generation in BAL cell membrane were determined (n = 5). (E) Lung colony-forming units (n = 5) and (F) Kaplan-Meier survival curves were determined (n = 6–7). **P < 0.001; ***P < 0.0001. Values shown represent means ± SEM. Two-tailed Student’s t-test statistical analysis was used for A and E, one-way ANOVA with Tukey statistical analysis was used for B and D, and a log-rank (Mantel-Cox) test for F. Results were repeated two times for A. Results were repeated three times for B–F, with a representative immunoblot shown. BALF = BAL fluid; ns = not significant.

Figure 5.

Cigarette smoke extract (CSE) and cadmium alter Rac2 (Ras-related C3 botulinum toxin substrate 2) isoprenylation. (A) Schematic diagram of full-length Rac2_WT construct showing location of cysteine residues and Rac2 construct with Cys (C)→Ser (S) mutation at amino acid 189. (B) MH-S cells expressing empty or Flag-Rac2_WT were treated with vehicle, CdCl₂ (80 μM, 3 h), or CSE and were separated into aqueous or detergent fractions. Immunoblot analysis was performed. (C) MH-S cells expressing empty or Flag-Rac2_C189S were treated with vehicle, CdCl₂, or CSE and were separated into aqueous or detergent fractions. Immunoblot analysis was performed. (D) Macrophages expressing empty, Flag-Rac2_WT, or Flag-Rac2_C189S were treated with vehicle, CdCl₂, or CSE. Immunoblot analysis was performed in MH-S cells in isolated membrane fraction. MH-S cells coexpressing empty and YFP-Rac2_CA, or Flag-Rac2_C189S, or YFP-Rac2_CA and Flag-Rac2_C189S were treated with vehicle or CdCl₂. Quantification of immunoblot analysis was performed in isolated aqueous or detergent fractions for (E) GFP-Rac2 (n = 3) and (F) Flag-Rac2 (n = 3). (G) Histidine (His) pull-down in MH-S cells coexpressing empty or p67^phox-V5-His together with Flag-Rac2_WT or Flag-Rac2_C189S treated with vehicle or CdCl₂. ***P < 0.0001. Values shown represent means ± SEM. One-way ANOVA with Tukey statistical analysis was used for E and F. Results were repeated three times for all data in B–D and G, with representative immunoblots shown. A = aqueous; D = detergent; IB = immunoblot; PD = pull-down; V = vehicle; WT = wild type.

Figure 6.

Rac2 (Ras-related C3 botulinum toxin substrate 2) activity is reduced in BAL cells from smokers. BAL was obtained from nonsmoking and smoking subjects (n = 4–6). (A) Cell differential was determined from BAL cells (n = 4–6). (B) Inductively coupled plasma mass spectrometry was performed for the indicated metals from BAL fluid (n = 4–6). (C) Rac2 activity in BAL cell membrane (n = 4–6). Immunoblot analysis of BAL cells was performed in (D) isolated membrane and (E) cytosol fractions. (F) NOX2 (NADPH oxidase 2)-derived reactive oxygen species generation in BAL cell membrane (n = 4). *P < 0.05; ***P < 0.0001. Values shown represent means ± SEM. One-way ANOVA with Tukey statistical analysis was used for A, and two-tailed Student’s t-test statistical analysis was used for B, C, and F. Results are from one experiment with n = 4–6 subjects. BALF = BAL fluid; Lymph = lymphocyte; Mac = macrophage/monocyte; ns = not significant; PMN = polymorphonuclear neutrophil.

Figure 7.

Rac2 (Ras-related C3 botulinum toxin substrate 2) ameliorates cigarette smoke (CS)–mediated lung infection. Wild-type mice were administered pLVX-IRES-tdTomato (empty) or pLVX-IRES-tdTomato-Rac2_CA (Rac2_CA) lentivirus. (A) Representative confocal images of CD11c-, Ly6c-, and RFP-positive BAL cells counterstained with DAPI. Scale bar, 25 μm (n = 3). Wild-type mice were transduced with empty or Rac2_CA lentivirus. After 6 weeks, mice were exposed to air or CS and then infected with saline or Streptococcus pneumoniae. (B) Immunoblot analysis of isolated BAL cell membrane. (C) Immunoblot analysis of isolated BAL cell membrane and cytosol with corresponding (D) Rac2 activity in BAL cell membrane (n = 4–8). (E) NOX2 (NADPH oxidase 2)-derived reactive oxygen species generation in BAL cell membrane (n = 5). (F) Lung colony-forming units (cfu; n = 5). Hematoxylin and eosin staining was performed in (G) empty-transduced (n = 5) and (H) Rac2_CA-transduced wild-type mice (n = 5). Scale bars, 600 μm. The second and fourth rows in G and H show magnifications of the boxed regions in the first and third rows, respectively. *P < 0.05; ***P < 0.0001. One-way ANOVA with Tukey statistical analysis were used for D–F. Representative immunoblots and histology micrographs are shown. Mem = membrane; OD = optical density; RFP = red fluorescent protein; S.p. = Streptococcus pneumoniae.

Figure 8.

Lung macrophage Rac2 (Ras-related C3 botulinum toxin substrate 2) promotes macrophage host defense. Rac2^−/− mice were administered pLVX-IRES-tdTomato (empty) or pLVX-IRES-tdTomato-Rac2_CA (Rac2_CA) lentivirus (5 × 10⁷ TU, intratracheally). Six weeks later mice were exposed to saline or Streptococcus pneumoniae. (A) Rac2 activity in BAL cell membrane from Rac2^−/− mice (n = 3–5). (B) NOX2 (NADPH oxidase 2)-derived reactive oxygen species generation in BAL cell membrane was determined (n = 5). (C) Lung colony-forming units (cfu; n = 5) and (D) Kaplan-Meier survival curves were performed (n = 7–9). **P < 0.001; ***P < 0.0001. Values shown represent means ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test was used for A and B, two-tailed t-test statistical analysis was used for C, and a log-rank (Mantel-Cox) test was used for D. OD = optical density; S.p. = Streptococcus pneumoniae.