Original Article
Doxycycline suppresses Chlamydia pneumoniae induced interferon-gamma responses in peripheral blood mononuclear cells in children with allergic asthma

https://doi.org/10.1016/j.jiac.2018.02.004Get rights and content

Abstract

Persistent respiratory infections caused by Chlamydia pneumoniae have been implicated in the pathogenesis of chronic diseases (e.g. asthma). Antibiotics are used to treat C. pneumoniae respiratory infections; however, the use of antibiotics as anti-inflammatory agents in treatment of asthma remains controversial. The current study investigated whether ciprofloxacin, azithromycin, or doxycycline can suppress C. pneumoniae-induced production of immunoglobulin (Ig) E or cytokines in peripheral blood mononuclear cells (PBMC) obtained from asthmatic children. Apart from blood, nasopharyngeal swab specimens were also collected to test for the presence of C. pneumoniae and/or M. pneumoniae (qPCR). PBMC (1.5 x 106) from asthmatic pediatric patients (N = 18) were infected or mock infected for 1 h ± C. pneumoniae AR-39 at a multiplicity of infection (MOI) = 0.1, and cultured ± ciprofloxacin, azithromycin, or doxycycline (0.1 or 1.0 μg/mLmL) for either 48 h (cytokines) or 10 days (IgE). Interleukin (IL)-4, interferon (IFN)-γ and IgE levels in supernatants were measured (ELISA). When PBMC were infected with C. pneumoniae, IL-4 and IFNγ production increased (p = 0.06 and 0.03, respectively); IgE levels were low. The now-elevated levels of IL-4 didn't decrease significantly after addition of ciprofloxacin, azithromycin, or doxycycline. However, infected PBMC IFNγ formation decreased significantly when 0.1 μg/mL doxycycline was employed (p = 0.04); no dose of ciprofloxacin or azithromycin had any impact. This inhibitory outcome with doxycycline lends support to the use of tetracyclines as immune modulators and anti-inflammatory medications in treatment of C. pneumoniae-infected asthma patients.

Introduction

Chlamydia pneumoniae is an obligate intracellular organism [1] that causes respiratory infections in adults and children and has been implicated in the pathogenesis of chronic disease (e.g. asthma) [2]. Prior studies have shown that C. pneumoniae can cause prolonged respiratory infection in asthmatic and non-asthmatic subjects [3], [4], [5]. Infection with C. pneumoniae can activate immune cells (e.g. macrophages, endothelial and epithelial cells) to produce cytokines that may contribute to the pathology observed in asthma [2]. In addition, C. pneumoniae infection triggers the production of pathogen-specific IgE in children with chronic respiratory disease, which may lead to inflammation [6].

Treatment of atypical bacteria, specifically C. pneumoniae and Mycoplasma pneumoniae, with antibiotics (macrolides, tetracyclines, quinolones) may have beneficial effects in patients with asthma because of the eradication of acute or persistent C. pneumonaie infection [2], [7], and potential pleiotropic anti-inflammatory properties in addition to anti-chlamydial activity.

Previous studies in our laboratory investigated the steroid-sparing effect of the tetracycline, minocycline, when given to adults with moderate persistent or severe persistent asthma, in addition to steroid treatment [8]. Minocycline treatment was associated with improvement in pulmonary function and asthma symptoms [8]; the effect was independent of the presence of C. pneumoniae infection (culture and serology) [8]. The observed effect may have been attributed to inflammation suppression or eradication of the C. pneumoniae [3], [9]. We have also previously demonstrated that doxycycline can suppress C. pneumoniae mediated increases in ongoing IgE and IL-4 responses by PBMC of patients with asthma [10]; the anti-inflammatory effects of doxycycline observed in that model, were highest in the presence of memory lymphocyte responses to C. pneumoniae [10]. It has been speculated that targeting inflammatory mediators (e.g. IgE production) may help improve asthma symptoms [10].

Quinolones (levofloxacin and moxifloxacin), azithromycin and doxycycline are antibiotics which are frequently used for the treatment of C. pneumoniae respiratory infections [7]. Ciprofloxacin is significantly less active than moxifloxacin and levofloxacin and is not used to treat C. pneumoniae respiratory infections in patients [7]; however, ciprofloxacin has been used in the in vitro setting to test drug class, as well as anti-inflammatory activity independent of antimicrobial activity [11].

The role of macrolide antibiotics in asthma is ongoing and the major areas of disagreement include the specific impact of these antibiotics. The present study describes whether the role of macrolide antibiotics is anti-inflammatory (having a non-specific effect) or bactericidal (leading to clearance of pathogens and resolution of asthma symptoms). This was achieved by studying the effect of ciprofloxacin, azithromycin, or doxycycline on C. pneumoniae induced cytokine responses (interferon [IFN]-γ and IL-4) in children with allergic asthma, as well as the ability of these drugs to regulate in vitro IgE responses.

Section snippets

Study design

We recruited pediatric patients with allergic asthma (male/female, 8–20 years old) from the outpatient department at SUNY Downstate Medical Center (Brooklyn, NY). Inclusion criteria included a physician's diagnosis of stable asthma without current respiratory infection, or current clinically defined persistent asthma symptoms [12], or both, with elevated serum IgE levels (>100 IU/mL). Asthma diagnosis was made at least one year before study enrollment. Exclusion criteria included a history of

Study population demographics

A total of 18 pediatric asthmatic patients (9 male, 9 female; mean age = 13 [± 4] years [range 8–20 years]) were enrolled in the study. All were classified as having moderate persistent asthma and all had been treated with inhaled corticosteroids. Inhaled corticosteroids included Budesonide 500 mcg or Fluticasone HFA (176, 220 or 440 mcg). Leukotriene modifiers included Monteleukast (5 or 10 mg). Total serum IgE levels were high in the patient population (665 [± 159] IU/mL). Table 1 shows the

Discussion

The current study examined in vitro activities of ciprofloxacin, azithromycin, and doxycycline in C. pneumoniae-infected PBMC, as well as their ability to regulate cytokine (IFNγ and IL-4) and IgE responses. The results demonstrated that in this ex-vivo PBMC model: (1) only doxycycline (at 0.1 μg/mL) suppressed IFNγ production, (2) no drug had a significant effect on IL-4 production, and (3) none of the three drugs affected IgE production. The results here may assist in antibiotic selection for

Conclusions

The findings presented in this study highlight for the first time, to our knowledge, the suppressive effect of doxycycline on C. pneumoniae induced IFN-γ responses in PBMC from children with allergic asthma. Drug trials evaluating antibiotics with potential anti-inflammatory effects in patients with asthma need to be based on pre-clinical studies identifying the optimal drug class and concentration for optimal impact, as potentially effective strategies for the treatment of asthma.

Ethics and consent to participate

The SUNY Downstate Medical Center Institutional Review Board (IRB) approved this study with the need for written informed consent to participate in the study.

Consent to publish

Not applicable. This manuscript does not contain any individual persons' data.

Competing interests

All authors declare no financial and non-financial competing interests.

Funding source

This work was supported, in part by the Thrasher Research Fund (Young Investigator Award to Stephan Kohlhoff, M.D.).

Transparency declaration

None to declare.

Acknowledgements

We would like to thank Kevin Norowitz, M.D. (SUNY Downstate Med Center, Dept. of Pediatrics) for fruitful discussions.

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