Impact of Cigarette Smoke Exposure on Innate Immunity: A Caenorhabditis elegans Model

BACKGROUND: Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and lung cancer. Respiratory bacterial infections have been shown to be involved in the development of COPD along with impaired airway innate immunity. METHODOLOGY/PRINCIPAL FINDINGS: To address the in vivo impact of cigarette smoke (CS) exclusively on host innate defense mechanisms, we took advantage of Caenorhabditis elegans (C. elegans), which has an innate immune system but lacks adaptive immune function. Pseudomonas aeruginosa (PA) clearance from intestines of C. elegans was dampened by CS. Microarray analysis identified 6 candidate genes with a 2-fold or greater reduction after CS exposure, that have a human orthologue, and that may participate in innate immunity. To confirm a role of CS-down-regulated genes in the innate immune response to PA, RNA interference (RNAi) by feeding was carried out in C. elegans to inhibit the gene of interest, followed by PA infection to determine if the gene affected innate immunity. Inhibition of lbp-7, which encodes a lipid binding protein, resulted in increased levels of intestinal PA. Primary human bronchial epithelial cells were shown to express mRNA of human Fatty Acid Binding Protein 5 (FABP-5), the human orthologue of lpb-7. Interestingly, FABP-5 mRNA levels from human smokers with COPD were significantly lower (p = 0.036) than those from smokers without COPD. Furthermore, FABP-5 mRNA levels were up-regulated (7-fold) after bacterial (i.e., Mycoplasma pneumoniae) infection in primary human bronchial epithelial cell culture (air-liquid interface culture). CONCLUSIONS: Our results suggest that the C. elegans model offers a novel in vivo approach to specifically study innate immune deficiencies resulting from exposure to cigarette smoke, and that results from the nematode may provide insight into human airway epithelial cell biology and cigarette smoke exposure

Electronic cigarette liquid increases inflammation and virus infection in primary human airway epithelial cells.

The use of electronic cigarettes (e-cigarettes) is rapidly increasing in the United States, especially among young people since e-cigarettes have been perceived as a safer alternative to conventional tobacco cigarettes. However, the scientific evidence regarding the human health effects of e-cigarettes on the lung is extremely limited. The major goal of our current study is to determine if e-cigarette use alters human young subject airway epithelial functions such as inflammatory response and innate immune defense against respiratory viral (i.e., human rhinovirus, HRV) infection.We examined the effects of e-cigarette liquid (e-liquid) on pro-inflammatory cytokine (e.g., IL-6) production, HRV infection and host defense molecules (e.g., short palate, lung, and nasal epithelium clone 1, SPLUNC1) in primary human airway epithelial cells from young healthy non-smokers. Additionally, we examined the role of SPLUNC1 in lung defense against HRV infection using a SPLUNC1 knockout mouse model. We found that nicotine-free e-liquid promoted IL-6 production and HRV infection. Addition of nicotine into e-liquid further amplified the effects of nicotine-free e-liquid. Moreover, SPLUNC1 deficiency in mice significantly increased lung HRV loads. E-liquid inhibited SPLUNC1 expression in primary human airway epithelial cells. These findings strongly suggest the deleterious health effects of e-cigarettes in the airways of young people. Our data will guide future studies to evaluate the impact of e-cigarettes on lung health in human populations, and help inform the public about potential health risks of e-cigarettes

E-liquid induces IL-6 production in primary human airway epithelial cells.

<p>Normal human tracheobronchial epithelial cells from young healthy non-smokers were exposed to medium (control), e-liquid at various concentrations without nicotine (NO-NIC) or with 18 mg/ml of nicotine (NIC-18 mg/ml) for 24 h (A) and 48 h (B). IL-6 protein levels in cell culture supernatants were measured by ELISA. Data (n = 4) are presented as means ± SEM.</p

E-liquid inhibits SPLUNC1, a host defense molecule against human rhinovirus infection, in primary human airway epithelial cells.

<p>(A) SPLUNC1 knockout (KO, n = 5) mice and littermate controls (WT, n = 5) were intranasally inoculated with HRV-1B at 5×10⁶ pfu/mouse for 24 h to examine lung HRV RNA levels by quantitative real-time RT-PCR. Data are presented as means ± SEM. (B) Normal human tracheobronchial epithelial cells from young healthy non-smokers were pre-exposed to medium (control), e-liquid at an optimized concentration (0.3% v/v) without nicotine (NO-NIC) or with 18 mg/ml of nicotine (NIC-18 mg/ml) for 24 h and then infected with HRV-16 at 10⁴ TCID₅₀/well or PBS (control) for 6 h. SPLUNC1 mRNA levels were measured by quantitative real-time RT-PCR. Data (n = 5) are presented as means ± SEM.</p

E-liquid promotes human rhinovirus (HRV) infection in primary human airway epithelial cells.

<p>Normal human tracheobronchial epithelial cells from young healthy non-smokers were pre-exposed to medium (control), e-liquid at an optimized concentration (0.3% v/v) without nicotine (NO-NIC) or with 18 mg/ml of nicotine (NIC-18 mg/ml) for 24 h and then infected with HRV-16 at 10⁴ TCID₅₀/well or PBS (control) for 6 h (A) and 24 h (B). Viral RNA levels were measured by quantitative real-time RT-PCR. Data (n = 5) are presented as means ± SEM.</p

The physiological doses of e-liquid do not decrease primary human airway epithelial cell viability.

<p>Normal human tracheobronchial epithelial cells from young healthy non-smokers were treated with medium (control), e-liquid at various concentrations without nicotine (NO-NIC) or with 18 mg/ml of nicotine (NIC-18 mg/ml) for 24 h (A) and 48 h (B). Cell toxicity was assessed by measuring lactate dehydrogenase (LDH) levels in cell culture supernatants. Data (n = 4) are presented as mean ± SEM.</p

Airway epithelial NF-κB activation promotes Mycoplasma pneumoniae clearance in mice.

Respiratory infections including atypical bacteria Mycoplasma pneumoniae (Mp) contribute to the pathobiology of asthma and chronic obstructive pulmonary disease (COPD). Mp infection mainly targets airway epithelium and activates various signaling pathways such as nuclear factor κB (NF-κB). We have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1) serves as a novel host defense protein and is up-regulated upon Mp infection through NF-κB activation in cultured human and mouse primary airway epithelial cells. However, the in vivo role of airway epithelial NF-κB activation in host defense against Mp infection has not been investigated. In the current study, we investigated the effects of in vivo airway epithelial NF-κB activation on lung Mp clearance and its association with airway epithelial SPLUNC1 expression.Non-antimicrobial tetracycline analog 9-t-butyl doxycycline (9-TB) was initially optimized in mouse primary tracheal epithelial cell culture, and then utilized to induce in vivo airway epithelial specific NF-κB activation in conditional NF-κB transgenic mice (CC10-(CA)IKKβ) with or without Mp infection. Lung Mp load and inflammation were evaluated, and airway epithelial SPLUNC1 protein was examined by immunohistochemistry. We found that 9-TB treatment in NF-κB transgene positive (Tg+), but not transgene negative (Tg-) mice significantly reduced lung Mp load. Moreover, 9-TB increased airway epithelial SPLUNC1 protein expression in NF-κB Tg+ mice.By using the non-antimicrobial 9-TB, our study demonstrates that in vivo airway epithelial NF-κB activation promotes lung bacterial clearance, which is accompanied by increased epithelial SPLUNC1 expression

In vivo function of airway epithelial TLR2 in host defense against bacterial infection

Decreased Toll-like receptor 2 (TLR2) expression has been reported in patients with chronic obstructive pulmonary disease and in a murine asthma model, which may predispose the hosts to bacterial infections, leading to disease exacerbations. Since airway epithelial cells serve as the first line of respiratory mucosal defense, the present study aimed to reveal the role of airway epithelial TLR2 signaling to lung bacterial [i.e., Mycoplasma pneumoniae (Mp)] clearance. In vivo TLR2 gene transfer via intranasal inoculation of adenoviral vector was performed to reconstitute TLR2 expression in airway epithelium of TLR2−/− BALB/c mice, with or without ensuing Mp infection. TLR2 and lactotransferrin (LTF) expression in airway epithelial cells and lung Mp load were assessed. Adenovirus-mediated TLR2 gene transfer to airway epithelial cells of TLR2−/− mice reconstituted 30–40% TLR2 expression compared with TLR2+/+ cells. Such airway epithelial TLR2 reconstitution in TLR2−/− mice significantly reduced lung Mp load (an appropriate 45% reduction), coupled with elevated LTF expression. LTF expression in mice was shown to be mainly dependent on TLR2 signaling in response to Mp infection. Exogenous human LTF protein dose-dependently decreased lung bacterial load in Mp-infected TLR2−/− mice. In addition, human LTF protein directly dose-dependently decreased Mp levels in vitro. These data indicate that reconstitution of airway epithelial TLR2 signaling in TLR2−/− mice significantly restores lung defense against bacteria (e.g., Mp) via increased lung antimicrobial protein LTF production. Our findings may offer a deliverable approach to attenuate bacterial infections in airways of asthma or chronic obstructive pulmonary disease patients with impaired TLR2 function

9-TB treatment increases KC and IL-6 levels in CC10-_CAIKKβ transgene positive (Tg+), but not transgene negative (Tg–) mice with saline treatment.

<p>(<b>A</b>) – KC; (<b>B</b>) – IL-6. N = 4–6 mice per group. Data are expressed as means ± SEM.</p

9-TB enhances NF-κB activation in saline-treated CC10-_CAIKKβ Tg+ mice.

<p>NF-κB activity in 9-TB- and saline-treated CC10-_CAIKKβ Tg+ mice was measured by using the NF-κB p65 ELISA in nuclear proteins extracted from mouse lungs (n = 4 mice per group). Data are expressed as means ± SEM.</p