Cigarette smoke inhibits BAFF expression and mucosal immunoglobulin A responses in the lung during influenza virus infection

Abstract

Background: It is incompletely understood how cigarette smoke (CS) exposure affects lung mucosal immune responses during viral respiratory infections. B cell activating factor belonging to the tumor necrosis factor family (BAFF) plays an important role in the induction of secretory immunoglobulin A (S-IgA) which is the main effector of the mucosal immune system. We therefore investigated the effects of CS exposure on BAFF expression and S-IgA responses in the lung during influenza virus infection.

Methods: Mice were exposed to CS and/or infected with influenza virus. Bronchoalveolar lavage fluid and lung compartments were analyzed for BAFF expression, influenza-specific S-IgA level and histological changes. Lung B cells were isolated and the activation-induced cytidine deaminase (Aicda) expression was determined. BEAS-2B cells were treated with CS extract (CSE), influenza virus, interferon beta or N-acetylcysteine and BAFF expression was measured.

Results: CS inhibited BAFF expression in the lung, particularly after long-term exposure. BAFF and S-IgA levels were increased during influenza virus infection. Three-month CS exposure prior to influenza virus infection resulted in reduced BAFF and S-IgA levels in the lung as well as augmented pulmonary inflammation on day 7 after infection. Prior CS exposure also caused decreased Aicda expression in lung B cells during infection. Neutralization of BAFF in the lung resulted in reduced S-IgA levels during influenza virus infection. CSE inhibited virus-mediated BAFF induction in a dose-dependent manner in BEAS-2B cells, while this inhibition of BAFF by CSE was prevented by pretreatment with the antioxidant N-acetylcysteine.

Conclusions: Our findings indicate that CS may hinder early mucosal IgA responses in the lung during influenza virus infection through oxidative inhibition of BAFF, which might contribute to the increased incidence and severity of viral infections in smokers.

Figure 1

Cigarette smoke (CS) induces inflammation but inhibits BAFF expression in the lung. Mice were exposed to room air (RA) or CS for 1 month, 3 months or 6 months. The relative mRNA levels of (A) BAFF and (C) keratinocyte-derived chemokine (KC) in the lung, (B) BAFF protein levels and (D) the total leukocyte counts in bronchoalveolar lavage (BAL) fluid are shown (n = 5–10 mice/group). *P < 0.05; **P < 0.01.

Figure 2

Influenza virus induces BAFF expression and mucosal IgA responses in the lung. Mice were infected with influenza virus and sacrificed on day 1, day 7 and day 14 after infection. (A) The relative mRNA levels of keratinocyte-derived chemokine (KC) in the lung, (B) the total leukocyte counts, (C) BAFF protein levels and (D) influenza-specific secretory IgA (S-IgA) levels in bronchoalveolar lavage (BAL) fluid are shown (n = 3–5 mice/group). CTRL, controls; #, not detected; *P < 0.05; **P < 0.01.

Figure 3

Cigarette smoke (CS) exposure prior to influenza virus infection results in augmented lung inflammation. Mice were exposed to room air (RA) or CS for 3 months and then infected with influenza virus or vehicle control. They were sacrificed and evaluated on day 7 after infection. Representative haematoxylin and eosin stained lung sections from (A) RA-exposed mice, (B) CS-exposed mice, (C) RA-exposed mice with influenza virus infection and (D) CS-exposed mice with influenza virus infection are shown. Scale bar, 100 μm; Original magnification, ×100.

Figure 4

Cigarette smoke (CS) exposure prior to influenza infection results in reduced BAFF and secretory IgA (S-IgA). Mice were exposed to room air (RA) or CS for 3 months and then infected with influenza virus or vehicle control. They were sacrificed and evaluated on day 7 after infection. (A) The relative mRNA levels of keratinocyte-derived chemokine (KC) in the lung, (B) the total leukocyte counts, (C) BAFF protein levels and (D) influenza-specific S-IgA levels in bronchoalveolar lavage (BAL) fluid are shown (n = 5 mice/group). RA + Flu, RA-exposed mice with influenza virus infection; CS + Flu, CS-exposed mice with influenza virus infection; #, not detected; *P < 0.05.

Figure 5

Cigarette smoke (CS) exposure prior to influenza virus infection results in reduced BAFF expression. Mice were exposed to room air (RA) or CS for 3 months and then infected with influenza virus or vehicle control. They were sacrificed and evaluated on day 7 after infection. Representative immunohistofluorescence stained lung sections from (A) RA-exposed mice with influenza virus infection (cells in bronchial lumen), (B) CS-exposed mice with influenza virus infection (cells in bronchial lumen), (C) RA-exposed mice with influenza virus infection (bronchial epithelium) and (D) CS-exposed mice with influenza virus infection (bronchial epithelium) are shown (BAFF: red, nuclear stain DAPI: blue). Original magnification, ×200.

Figure 6

Cigarette smoke (CS) exposure prior to influenza infection causes decreased Aicda in lung B cells. Mice were exposed to room air (RA) or CS for 3 months and then infected with influenza virus or vehicle control. Lung B cells were isolated on day 7 after infection. The relative mRNA levels of (A) Aicda, (B) BAFF receptor (BAFF-R), (C) transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI) and (D) B cell maturation antigen (BCMA) in lung B cells are shown (n = 5 mice/group). RA + Flu, RA-exposed mice with influenza virus infection; CS + Flu, CS-exposed mice with influenza virus infection; *P < 0.05.

Figure 7

BAFF neutralization results in reduced IgA responses in the lung during influenza virus infection. Mice were intranasally administered with BAFF-R-Fc, isotype control or PBS and infected with influenza virus. They were sacrificed and evaluated on day 7 after infection. (A) The influenza-specific secretory IgA (S-IgA) levels in bronchoalveolar lavage (BAL) fluid, (B) the relative mRNA levels of keratinocyte-derived chemokine (KC) in the lung, (C) the total leukocyte counts in BAL fluid and (D) the relative mRNA levels of Aicda in lung B cells are shown (n = 5 mice/group). Flu, infected mice with treatment of PBS; Isotype + Flu, infected mice with treatment of isotype control; BAFF-R-Fc + Flu, infected mice with treatment of BAFF-R-Fc; *P < 0.05.

Figure 8

Antioxidant N-acetylcysteine (NAC) prevents the inhibition of virus-mediated BAFF induction by cigarette smoke extract (CSE). (A) BEAS-2B cells were treated with indicated concentrations of CSE and/or infected with influenza virus at a multiplicity of infection (MOI) of 0.5 for 24 hours. (B) BEAS-2B cells were treated with indicated concentrations of interferon beta (IFN-β) for 24 hours. (C) BEAS-2B cells were treated with indicated concentrations of IFN-β in the combination with 5% CSE and influenza virus at 0.5 MOI for 24 hours. (D) BEAS-2B cells were pretreated with indicated concentrations of N-acetylcysteine (NAC) for 2 hours and then treated with 5% CSE and influenza virus at 0.5 MOI for 24 hours. The relative mRNA levels of BAFF in BEAS-2B cells are shown. CTRL, without influenza virus infection; Flu, with influenza virus infection; Flu + CSE, with CSE treatment and influenza virus infection; NS, nonsignificant; *P < 0.05; **P < 0.01.