INCREASED COTININE CONCENTRATIONS ARE ASSOCIATED WITH REDUCED EXPRESSION OF CATHELICIDIN (LL-37) AND NOD-2 in ALVEOLAR MACROPHAGES OF PLWH WHO SMOKE

Abstract

There is a strong link between cigarette smoking and pulmonary complications among people living with HIV. However, the effects of smoking on the local lung immune environment in this population remain unclear. Bronchoalveolar lavage and saliva were collected from HIV-infected smokers involved in a prospective study investigating alveolar macrophage expression of host defense molecules. Salivary cotinine concentrations were inversely related to expression of the immune cell receptor NOD-2 and the cathelicidin antimicrobial peptide LL-37. The negative correlation between salivary cotinine and LL-37 was particularly strong. Our study provides insight into how nicotine may adversely affect lung innate immunity in HIV.

Introduction

The prevalence of tobacco use among people living with HIV (PLWH) is two to threefold higher than that of the general population [1] and there is a prominent link between a number of important HIV-related pulmonary complications and cigarette smoking. For example, current smoking status is among the most important risk factors for development of lower respiratory tract infections in this group [2] and PLWH who smoke are unusually susceptible to the development of chronic obstructive pulmonary disease (COPD) [3]. It has been hypothesized that lower respiratory tract infections or colonization of microorganisms may contribute to the development or progression of air-flow limiting diseases among PLWH who smoke [3].

There remains limited information on the effects of smoking on the local lung immune and inflammatory environment in this population. In particular, the effect of smoking on a number of important host defense molecules including Toll-like receptors (TLRs) Nod-like receptors (NLRs), and the human cathelicidin antimicrobial peptide LL-37 has not been explored. In this study, we examined alveolar macrophage gene expression of a number of host-defense molecules as well as number of cytokines potentially relevant to HIV-related pulmonary disease in a group of PLWH who smoke. The primary objective was to determine whether there was a relationship between the degree of smoke exposure and the expression of these molecules in the lung.

Methods

Study Participants:

Subjects in this study were part of a larger prospective study conducted at The Ohio State University (OSU) examining a smoking cessation intervention for HIV-infected smokers (n = 262). The OSU participants in this study were recruited between 2008–2013, as one site of the Lung HIV study, a multisite study funded by the National Heart, Lung and Blood Institute for the purpose of better understanding longitudinal outcomes and pulmonary complications among PLWH. Eligibility criteria for the Ohio State study included HIV positive status, daily smoking (at least 5 cigarettes per day), and an interest in quitting smoking in the next 30 days. The subset involved in this investigation were those individuals (n = 71) who agreed to participate in OSU’s bronchoalveolar lavage (BAL) sub-study examining the effects of smoking on alveolar macrophage biology.

The clinical study procedures were performed primarily in the OSU Pulmonary Clinical Research Unit and the OSU Clinical Research Center. Studies were approved by the Ohio State University IRB and informed consent was obtained on all study subjects.

Salivary cotinine.

Participants’ saliva was collected in cryotubes which were stored at −70 C and then sent to J2 Laboratories, (Tuscon Az). Cotinine levels were determined using gas chromatography-mass spectrometry.

Alveolar macrophage gene expression:

We measured mRNA levels of the human cathelicidin antimicrobial peptide LL-37 (CAMP gene), immune cell receptors (TLR-2, TLR-4, NOD-1, NOD-2) and a number of cytokines potentially linked to HIV and/or COPD pathogenesis (IL-1β, IL-6, IL-8, IL-10, IL-18, IFN-g, TNFα). After informed consent subjects underwent standard bronchoscopy with BAL consisting of sequentially instilling and aspirating sterile saline in seven 20 ml aliquots in the right middle lobe bronchus from the wedged position. Recovered fluid was passed through a single layer of surgical gauze to remove mucus and particulate matter. Fluid aliquots were taken for cell counting. Macrophages were recovered and lysed in Trizol reagent (Invitrogen Life Technologies) for RNA purification. Approximately 2–3 ug of total RNA was converted to cDNA via ThermoScript reverse transcriptase (Invitrogen Life Technologies) with oligo dT primers.

Real Time PCR:

Macrophage mRNA levels were quantified by real time PCR using Syber green and primers designed to allow multiple gene comparisons within the same run as we have previously described [4]. The Ct of target genes were normalized to the average Ct of two housekeeping genes GAPDH and CAP1 and expressed as relative copy number (RCN) (4)

Statistical Methods:

Descriptive statistics were calculated for demographic variables. To determine if there was an association between smoking intensity (as assessed by either salivary cotinine levels or self-reported cigarettes per day) and alveolar macrophage gene expression, linear regression models were fit for each comparison, with RT-qPCR data as the outcome and cotinine (or cigarettes per day) as the primary predictor. The models also controlled for age, gender, and CD4 count. Because of skewness of the data (except for TLR2 and TLR-4), RT-qPCR results were log transformed prior to running the linear regression models. After transformation, the residuals from the regression models were closer to being normally distributed and having equal variances.

Results

Baseline clinical characteristics of the study population:

The table demonstrates the baseline characteristics of the study population. The sex distribution of the population was similar to that of the HIV demographics of Central Ohio 12 females (16.9%) and 59 males (83.1%). The mean age of the participants was 42.3 years, 83% were male and 62% identified as Caucasian. Approximately three quarters of the subjects were on ART at the time of the study. Mean salivary cotinine was 230 ng/ml +/− 177

Alveolar macrophage gene expression and smoking intensity.

There was a strong inverse relationship between cotinine and macrophage gene expression of NOD-2 and LL-37. When controlling for age, gender and CD4 count, increasing cotinine levels were significantly associated with decreased expression of both NOD-2 (p = 0.009; R = 0.30) and LL-37 (p = 0.0004; R = 0.44). The figure demonstrates the scatter plots and fitted regression lines examining the relationship between cotinine concentrations and natural log of NOD-2 and LL-37 respectively. Interestingly, there was no relationship between subject reported cigarettes smoked per day and gene expression of either NOD-2 or LL-37. There was a weakly positive relationship between cotinine and macrophage gene expression of IL-1β and 1L-8. When controlling for age, gender and CD4 count, increasing cotinine levels were weakly associated with increased expression of both IL-1β (R = 0.26; p = 0.034) and IL-8 (R = 0.21; p = 0.029). There was no relationship between subject reported cigarettes smoked per day and gene expression of either IL-1β or IL-8.

There was no significant relationship between cotinine concentrations or patient reported cigarettes per day and alveolar macrophage gene expression of the other immune cell receptors or cytokines studied.

Discussion

Among PLWH cigarette smoking is one of the most important risk factors for the development of lower respiratory tract infections. For example, Garrido and colleagues recently reported the risk factors for invasive pneumococcal disease and community-acquired pneumonia (CAP) in a cohort of PLWH in a Dutch HIV referral center and found that current smoking was associated with a nearly three-fold risk for the development of CAP [2]. Furthermore, smokers with HIV appear to be at increased risk for smoking-related lung and airway injury, as HIV appears to be an independent risk factor for air-flow limiting disease. In a recent metaanalysis, global prevalence of COPD among PLWH was found to be high and COPD risk increased with income level and the presence of detectable viral load. As expected, smoking increased the risk but HIV remained an independent risk factor for airflow-limiting disease even after accounting for smoking history [3]. Notably, PJP as well as bacterial pneumonia predict subsequent progression of airflow obstruction and pulmonary diffusion impairment, supporting a connection between the risk of respiratory infection and airflow-limiting disease among PLWH [5]. Despite the importance of cigarette smoking as a risk factor for pulmonary complications, there is relatively little information regarding the effects of smoking on the local immune and inflammatory environment of the lung among PLWH. Our study provides insight into how smoking may affect lung innate immunity in HIV, as the nicotine metabolite cotinine, is inversely related to alveolar macrophage expression of both NOD-2 and LL-37.

Nucleotide-binding oligomerization domain (NOD)-2 is a member of the NOD-like family of intracellular pathogen receptors that are increasing being recognized for their role in innate immunity and the pathogenesis of chronic inflammatory states [6].The recognition by cytosolic NOD-2 receptors of the bacterial motif muramyl-dipeptide (MDP) activates NFkB signal cascade that ultimately leads to cytokine and chemokine transcription [6]. NOD-2 is expressed on professional antigen presenting cells (APC) and is present in the airway epithelium [6]. NOD-2 appears to have a role in the host response to several respiratory pathogens, including S. pneumonia, M. tuberculosis [7], L. pneumophilia [6] and it is possible that reduced expression of this immune cell receptor may contribute to the increased risk of respiratory infections among HIV-infected smokers.

The human cathelicidin antimicrobial peptide LL-37 is directly regulated by vitamin D [8], and is produced by monocytes, neutrophils, and epithelial cells. It is an integral part of the innate lung immunity [9–11]. LL-37 appears to plays a multifactorial role in host defense, acting as a “fire alarm” in the setting of infection - signaling an overwhelming threat and recruiting other host defense cells to target the infection [11]. Recent data suggest that deficiencies of LL-37 may be important in COPD pathobiology. In a cross-sectional analysis of a population with a high HIV prevalence (41%), plasma LL37 levels were independently associated with low FEV1 [12]. More recently, in a longitudinal analysis of current and former smokers, low plasma LL-37 predicted persistent lung function declines at six and 18 months [13]. While the pathogenesis of accelerated airflow-limiting disease in the setting of HIV remains unclear, it has been postulated that recurrent respiratory infections or chronic colonization may trigger an abnormal inflammatory or repair process in the lung and airways of HIV-infected smokers [3].

The significance of our findings showing a positive correlation between cotinine and increased expression of IL-β and IL-8 is unclear, as prior studies in HIV-negative subjects suggest that smoking decreases alveolar macrophage expression of IL-β and IL-8. It is possible that inflammatory processes associated with HIV modulate alveolar macrophage expression of these cytokines.

To our knowledge this study is the first in vivo investigation to measure the nicotine metabolite cotinine and correlate it with lung cell biology in humans. Our data demonstrate that cotinine concentrations, but not subject self-report of smoking amount, is significantly associated with macrophage expression of key immune molecules. Our data suggesting significant effects of nicotine on LL-37 dysregulation is supported by animal studies. In mice, activation of the cholinergic system by nicotinic receptors downregulates LL-37 in the skin and increases susceptibility to S. aureus skin infections [14]. The potential role of nicotine as a direct inhibitor on innate immunity in the lung is relevant as nicotine is increasingly being delivered via electronic cigarettes and recent studies have examined the use of electronic cigarettes as a smoking cessation tool among PLWH [15]. While suggested as a safer alternative to cigarette smoking [16], increasing concentrations of nicotine associated with vaping may have important adverse effects on lung immunity.

Our data demonstrate that among PLWH, the degree of cigarette smoke exposure affects alveolar macrophage gene expression of LL-37 and NOD-2, molecules important in the innate immune response. The linkage between LL-37 and cotinine levels may help us understand the heightened risk of HIV-infected smokers to develop lower respiratory tract infections as well as airflow-limiting disease.

Figure 1.

Scatterplot and fitted regression line of the association between (A) cotinine concentration and natural log of NOD-2 and (B) cotinine concentration and natural log of LL-37.

Table 1.

Demographic and clinical characteristics of HIV-infected participants (n=71)

Characteristic	N (%)	Mean ± SD
Age		42.3 ± 9.5
Male	59 (83.1%)
White Race	62%
Current cigarettes per day		20.3 ± 13.0
Pack-years of smoking		27.2 ± 21.9
Baseline Cotinine (ng/mL)		230 ± 177
CD4 Count (cells/mm3)		507 ± 269
On antiretroviral therapy	73.9%