Azithromycin decreases Chlamydia pneumoniae-mediated Interleukin-4 responses but not Immunoglobulin E responses

Background Chlamydia pneumoniae is an obligate intracellular bacterium that causes respiratory infection. There may exist an association between C. pneumoniae, asthma, and production of immunoglobulin (Ig) E responses in vitro. Interleukin (IL-4) is required for IgE production. Objective We previously demonstrated that doxycycline suppresses C. pneumoniae-induced production of IgE and IL-4 responses in peripheral blood mononuclear cells (PBMC) from asthmatic subjects. Whereas macrolides have anti-chlamydial activity, their effect on in vitro anti-inflammatory (IgE) and IL-4 responses to C. pneumoniae have not been studied. Methods PBMC from IgE- adult atopic subjects (N = 5) were infected +/- C. pneumoniae BAL69, +/- azithromycin (0.1, 1.0 ug/mL) for 10 days. IL-4 and IgE levels were determined in supernatants by ELISA. IL-4 and IgE were detected in supernatants of PBMC (day 10). Results When azithromycin (0.1, 1.0 ug/ml) was added, IL-4 levels decreased. At low dose, IgE levels increased and at high dose, IgE levels decreased. When PBMC were infected with C. pneumoniae, both IL-4 and IgE levels decreased. Addition of azithromycin (0.1, 1.0 ug/mL) decreased IL-4 levels and had no effect on IgE levels. Conclusions These findings indicate that azithromycin decreases IL-4 responses but has a bimodal effect on IgE responses in PBMC from atopic patients in vitro.

Introduction Chlamydia pneumoniae is an intracellular bacterium that infects humans and causes respiratory infections [1,2] in asthmatic and non-asthmatic subjects [2][3][4][5]. This bacterium activates immune cells (e.g. macrophages, epithelial cells); these cells produce cytokines that may contribute to asthma exacerbation [2]. In children with chronic respiratory disease, C. pneumoniae infection triggers the production of pathogen specific IgE, which may lead to inflammatory responses [6].
C. pneumoniae, can be treated with antibiotics (macrolides, tetracyclines, quinolones) that may have beneficial effects in patients with asthma [2,7]. Tetracyclines and macrolides have demonstrated anti-inflammatory activity independent of their antimicrobial activity [7,8].
Previous studies in our laboratory reported that treatment of asthmatic adults with minocycline had improvement in their asthma symptoms, independent of the presence of C. pneumoniae infection and may be due to past exposure [11]. This observed effect may be due to suppression of inflammation or eradication of the C. pneumoniae [3,12].
Other studies in our laboratory demonstrated that doxycycline suppressed C. pneumoniaeinduced IgE and IL-4 responses in PBMC obtained from patients with asthma [13]. Smith-Norowitz et al recently reported that doxycycline suppressed C. pneumoniae induced interferon-gamma responses in PBMC in asthmatic children [14]. However, IL-4 levels did not significantly decrease after addition of ciprofloxacin (0.1 μg/ml) or azithromycin (1.0 μg/ml); C. pneumoniae infection and/or antibiotic treatment had no effect on IgE production in vitro [14].
The present study describes the in vitro effect of azithromycin on C. pneumoniae mediated IL-4 (Th2-type) cytokine responses and IgE responses in non-asthmatic atopic adults.

Study design
Adult subjects (male/ female, 18 to 65 years old) were recruited from the outpatient department at SUNY Downstate Medical Center (Brooklyn, NY). Inclusion criteria included: nonasthmatic adult with atopy (defined by a single unequivocal positive skin test or history of atopic dermatitis or allergic rhinitis) without clinically defined persistent asthma symptoms [15], with low serum IgE levels (<100 IU/mL). Exclusion criteria included: history of chronic immunosuppressive or autoimmune disease, human immunodeficiency virus infection, cancer, antibiotic use, or immunotherapy, tobacco use within the past year, and incomplete follow-up. All subjects had a nasopharyngeal (NP) swab tested for C. pneumoniae and/or M. pneumoniae (determined by PCR), and peripheral blood (10mL) was collected. All clinical data was reviewed at the time of enrollment. The study was approved by the SUNY Downstate Medical Center Institutional Review Board (Brooklyn, NY). Written informed consent was obtained from all participants.

Immunoglobulin determination: Total serum IgE
Total serum IgE levels were determined in serum using the UniCap Total IgE fluoroenzyme immunoassay (Pharmacia and Upjohn Diagnostics, Freiburg, Germany) as previously described [14]. Tests were performed in the Clinical Diagnostic Laboratory at SUNY Downstate Medical Center (Brooklyn, NY).

Cell cultures
PBMC were separated from blood on a Ficoll-Paque (GE Healthcare, Sweden) gradient (density 1.077) and put into cell culture a previously described [14], at 37˚C in cRPMI medium in a humidified 5% CO 2 atmosphere for up to 10 days. Cell viability was determined at 0, 48 and 240 hrs (>98%, 95%, and 90%, respectively), in the absence of any infection with C. pneumoniae.

In vitro infection with C. pneumoniae and treatment with antibiotics
Following a 2 hr incubation to allow adherence, PBMC cultures were infected with C. pneumoniae (by adding purified EB for 1hr), or mock-infected (MI) and/or stimulated in the presence or absence of azithromycin (0.1 or 1.0 ug/mL) (Sigma) for either 48 hrs (IL-4) or up to 10 days (IgE) at 37˚C in cRPMI in a humidified 5% CO 2 atmosphere, as previously described [14]. All antibiotics were serially diluted (1:1, 1:2, 1:4, 1:10) [13] to determine optimal dose and kinetics [13], for suppression (for the purpose of cytokine production). Cytokine assays (IL-4) were run using supernatants collected from above cultures. The multiplicity of infection (MOI; 0.1) and time points (48h p.i. for cytokines 10 and 10d p.i. for IgE 10 ) used for analysis were selected by kinetic and dose response studies (using MOI of 0.01-10) for optimization of the assay. Two types of controls were used in infection experiments: identical volumes of heat-inactivated purified C. pneumoniae [13] and identical volumes of HEp-2 cell cultures not containing any bacteria processed the same way as the purified C. pneumoniae [17] based on doseresponse experiments.

Cytokine (IL-4) or IgE determination: ELISA
For the in vitro quantitative determination of human cytokine or IgE content in cell culture supernatants, solid-phase sandwich ELISA assays were performed using either cytokine (IL-4: IL-4 Human ELISA kit, Thermo Fisher Scientific, Waltham, MA) or IgE ELISA test kits (Bio Quant, San Diego, CA), according to the manufacturer's recommended procedure, as previously described [14]. Cell culture supernatants were collected at either 48 hr p.i. (cytokines) [10] or 10 days p.i. (IgE) [10], by centrifugation, and samples were stored at -80˚until analysis. Optical densities were converted to either IU/mL, ng/mL, or pg/ml (1 IU IgE = 2.4 ng/IgE protein). Detection limits for cytokine assay was: IL-4: <2.0 pg/mL.

Quantitative real-time polymerase chain reaction (qPCR) of bacteria in swabs and cultures
Extractions of bacterial DNA from NP swab specimens [18] and PBMC were performed using a QIAAmp DNA Mini-Kit (Qiagen Inc., Valencia, CA), according to manufacturer's recommendations. For PBMC cultures, supernatants (with adherent and non-adherent cells) were collected and bacterial DNA extracted. NP samples were stored in the freezer (-20˚C), until analysis. DNA was extracted (QIAAmp DNA mini kit;Qiagen Inc), as previously described [18,19]. Specimens were tested for the presence and quantification of C. pneumoniae and M. pneumoniae DNA according to Apfalter et al. [18] and Waring et al. [19], using TAQ-Man technology-based qPCR (Light Cycler 2.0 platform; software version 4.0, Roche Diagnostics Corp, Indianapolis, IN). A specimen from either nostril positive for either C. pneumoniae or M. pneumoniae to represent a positive result.

Statistical analysis
Data are expressed as means ± SD unless elsewise indicated. Data between cases were analyzed using the Mann-Whitney U test. A P value of <0.05 was considered significant. All statistical analyses were performed using Windows v.12.0 software (SPSS Inc., Chicago, IL).

Study population demographics
Patient demographic and clinical characteristics is shown in Table 1. Five non-asthmatic atopic adult patients: 2 males (ages 24, 51) and 3 females (ages 51, 59, 65) were enrolled in this study. Total serum IgE levels were low (<100 IU/mL).

Serological and nucleic acid amplification testing for C. pneumoniae
Non-asthmatic patients had negative C. pneumoniae-specific IgG MIF titers. All patients tested negative for C. pneumoniae and M. pneumoniae by qPCR (NP swabs).

Effect of azithromycin on C. pneumoniae-induced IgE responses
When azithromycin (0.1 ug/mL) was added to cultures, IgE levels increased (50%), but when 1.0 ug/mL was added IgE levels decreased (38%) (Fig 2). When PBMC were infected with C.

Discussion
The effect of azithromycin on in vitro C. pneumoniae mediated cytokine and IgE responses and its contribution to inflammation are complex and not fully understood. In our initial report, we demonstrated that doxycycline suppresses C. pneumoniae mediated interferongamma responses in PBMC from children with asthma [14]; azithromycin did not decrease IL-4 or IgE responses [14]. The current study extends our prior study [14]; we further examine the in vitro effect of azithromycin on IL-4 and IgE response in C. pneumoniae-infected PBMC obtained from non-asthmatic adults. Successful treatment of C. pneumoniae infection with azithromycin may differ depending on asthma status. Our first finding was that addition of azithromycin decreased IL-4 responses in C. pneumoniae infected PBMC from non-asthmatic subjects. Recent studies of Smith-Norowitz et al reported that doxycycline can suppress in vitro C. pneumoniae mediated interferon-gamma responses in asthmatic children [14]. However, addition of ciprofloxacin (0.1 μg/ml) or azithromycin (1.0 μg/ml) did not decrease IL-4 responses [14]; in vitro IgE production was unaffected [14]. Earlier studies reported from our laboratory showed that suppression of C. pneumoniae-induced cytokine and IgE responses by doxycycline in vitro was independent of anti-chlamydial activity in PBMC from asthmatic patients [13]. Those findings suggest that in patients with asthma, antibiotics have an important anti-inflammatory contribution [13]. The studies differ most probably due to other factors or components of inflammation in asthmatic patients. The findings indicate that the in vitro effect of azithromycin in PBMC differs depending on asthma status. Thus, the relevance of these properties may be different based on asthma status as well as atopy status and needs to be considered for treatment guidelines.
We also found that treatment of cells with azithromycin could not suppress C. pneumoniaeinduced IgE production. The observed IgE production by azithromycin-treated infections might have been induced by other unknown contributors and not solely by C. pneumoniae infection. The IL-4 responses observed in our cultures represent a pathway for IgE production; it is possible that the failure of azithromycin to inhibit IgE responses was due to IL-4. The current findings are in agreement and supported by our previous studies [14] that reported azithromycin had no effect on C. pneumoniae-induced IgE responses in PBMC from patients with asthma [14].
The antibiotics azithromycin (macrolide), clarithromycin, and levofloxacin are used to treat C. pneumoniae respiratory infections in humans [20]. However, the recommended antibiotic doses may not be sufficient for C. pneumoniae infection eradication [20]. The current work extends our prior in vitro findings in asthmatic patients by examining the in vitro effect of azithromycin on C. pneumoniae mediated IL-4 and IgE responses in non-asthmatic atopic adults. Several important conclusions can be drawn from our data: (1) azithromycin decreases IL-4 responses but has a bimodal effect on IgE responses in PBMC from atopic patients in vitro and may have immunomodulatory properties; (2) at low concentrations, azithromycin does modulate C. pneumoniae-induced production of IL-4 but not IgE. Thus, depending on the concentration, azithromycin may inhibit production of proinflammatory mediators [10]; this may be independent of their antibacterial activities and due to the antimicrobiologic properties of azithromycin [10].
Some limitations to our study should be mentioned. We are aware of the small study number. Lastly, we did not elucidate the mechanism for the antibiotic effect on IL-4 production.