Human Immunology xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
Chlamydia pneumoniae enhances the Th2 profile of stimulated peripheral blood mononuclear cells from asthmatic patients Tamar A. Smith-Norowitz a,d,⇑, Kobkul Chotikanatis a, David P. Erstein b, Jason Perlman a, Yitzchok M. Norowitz a, Rauno Joks b,d, Helen G. Durkin c,d, Margaret R. Hammerschlag a, Stephan Kohlhoff a,d a
Department of Pediatrics, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States Department of Medicine, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States Department of Pathology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States d Center for Allergy and Asthma Research, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States b c
a r t i c l e
i n f o
Article history: Received 16 June 2015 Revised 23 February 2016 Accepted 24 February 2016 Available online xxxx Keywords: C. pneumoniae T helper cytokines IgE Asthma
a b s t r a c t Chlamydia pneumoniae is a cause of respiratory infection in adults and children. There is evidence for an association between atypical bacterial respiratory pathogens and the pathogenesis of asthma. We compared T helper (Th) responses in C. pneumoniae – infected peripheral blood mononuclear cells (PBMC) in patients with or without asthma. PBMC (1 106/mL) from asthmatic patients (N = 11) and non-asthmatic controls (N = 12) were infected or mock-infected for 1 h +/ C. pneumoniae TW-183 at a multiplicity of infection (MOI) = 1 and MOI = 0.1, or cultured for 24 h +/ Lactobacillus rhamnosus GG (LGG). Interleukin (IL)-4, IL-10, IL-12, Interferon (IFN)-gamma and total IgE levels were measured in supernatants (ELISA). C. pneumoniae infection led to an increase (>50%) of IgE levels in PBMC from asthmatics, compared with mock-infected on day 10; IgE wasn’t detected in non-asthmatics. C. pneumoniae – infected PBMC from asthmatics increased levels of IL-4 and IFN-gamma after 24 h, compared with PBMC alone; levels of IL-10 and IL-12 were low. When uninfected-PBMC from asthmatics were LGG-stimulated, after 24 h, IL-4 was undetectable, but IL-10, IL-12, and IFN-gamma increased, compared with PBMC alone. Thus, C. pneumoniae infection has the ability to induce allergic responses in PBMC of asthmatics, as evidenced by production of Th2 responses and IgE. Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction The importance of viral infections as infectious triggers of asthma exacerbations is well established [1,2]. The relationship between atypical bacterial infection (Chlamydia pneumoniae, Mycoplasma pneumoniae) and pathogenesis of asthma is a controversial topic [3]; studies investigating this association have been weak or provide conflicting data [4]. This may be due, in part, to inaccurate Abbreviations: PBMC, peripheral blood mononuclear cells; Abs, antibodies; Ig, Immunoglobulin; Th, T helper; TLR, toll like receptor; LGG, Lactobacillus rhamnosus GG; IL, Interleukin; AD, atopic dermatitis; MIF, microimmunofluorescence; MOI, multiplicity of infection; IFN, interferon; TNF, tumor necrosis factor; ELISA, enzyme linked immunosorbent assay; EB, elementary bodies; p.i., post-infection; TLR, tolllike receptor. ⇑ Corresponding author at: SUNY Downstate Medical Center, Dept of Pediatrics, Box 49, 450 Clarkson Ave., Brooklyn, NY 11203, United States. E-mail address: [email protected] (T.A. Smith-Norowitz).
diagnosis of infection [4], insensitive laboratory techniques [4], or poorly designed studies [4]. C. pneumoniae, an obligate intracellular bacterium [5], causes respiratory infection in adults and children and has been implicated in exacerbations of asthma [4]. Studies have shown that C. pneumoniae is capable of causing prolonged respiratory infections in asthmatic and non-asthmatics individuals [6–8]. Previous studies in our laboratory detected increased prevalence of anti-C. pneumoniae IgE Abs in children with wheezing compared with healthy controls and children with pneumonia who were not wheezing [9]. In addition, our laboratory demonstrated that doxycycline suppresses C. pneumoniae-mediated increases in ongoing IgE and IL-4 responses by peripheral blood mononuclear cells (PBMC) of patients with allergic asthma [10]. C. pneumoniae infection activates immune cells to produce cytokines that may contribute to the pathology seen in allergic
http://dx.doi.org/10.1016/j.humimm.2016.02.010 0198-8859/Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
Please cite this article in press as: T.A. Smith-Norowitz et al., Chlamydia pneumoniae enhances the Th2 profile of stimulated peripheral blood mononuclear cells from asthmatic patients, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.02.010
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T.A. Smith-Norowitz et al. / Human Immunology xxx (2016) xxx–xxx
asthma [4]. In addition, infection with C. pneumoniae triggers the production of pathogen-specific IgE in children with chronic respiratory disease, which may contribute to inflammation [11]. In the current study, we examined whether the presence of C. pneumoniae alters IgE levels and/or cytokine profiles in C. pneumoniae- infected PBMC from patients with asthma compared with non-asthmatic controls, using an in vitro model of C. pneumoniae infection and stimulation with Lactobacillus rhamnosus GG (LGG). LGG has been shown to modulate innate immune gene expression and attenuate inflammatory responses to pathogenic bacteria [12]. 2. Methods 2.1. Study participants Adult patients with allergic asthma (N = 11) and healthy nonasthmatic controls (N = 12) (male and female, 18–65 years old) were recruited from the outpatient department at SUNY Downstate Medical Center (Brooklyn, NY); written informed consent and assent was obtained from participants for use of their blood samples for an experimental study. Specific inclusion criteria for enrolled patients included a physician’s diagnosis of stable asthma or current clinically defined persistent asthma symptoms [13], or both, with elevated serum IgE levels (>100 IU/mL). Exclusion criteria included history of chronic immunosuppressive or autoimmune disease, human immunodeficiency virus infection, cancer, recent use of systemic corticosteroids (
Contents lists available at ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
Chlamydia pneumoniae enhances the Th2 profile of stimulated peripheral blood mononuclear cells from asthmatic patients Tamar A. Smith-Norowitz a,d,⇑, Kobkul Chotikanatis a, David P. Erstein b, Jason Perlman a, Yitzchok M. Norowitz a, Rauno Joks b,d, Helen G. Durkin c,d, Margaret R. Hammerschlag a, Stephan Kohlhoff a,d a
Department of Pediatrics, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States Department of Medicine, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States Department of Pathology, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States d Center for Allergy and Asthma Research, State University of New York Downstate Medical Center, Brooklyn, NY 11203, United States b c
a r t i c l e
i n f o
Article history: Received 16 June 2015 Revised 23 February 2016 Accepted 24 February 2016 Available online xxxx Keywords: C. pneumoniae T helper cytokines IgE Asthma
a b s t r a c t Chlamydia pneumoniae is a cause of respiratory infection in adults and children. There is evidence for an association between atypical bacterial respiratory pathogens and the pathogenesis of asthma. We compared T helper (Th) responses in C. pneumoniae – infected peripheral blood mononuclear cells (PBMC) in patients with or without asthma. PBMC (1 106/mL) from asthmatic patients (N = 11) and non-asthmatic controls (N = 12) were infected or mock-infected for 1 h +/ C. pneumoniae TW-183 at a multiplicity of infection (MOI) = 1 and MOI = 0.1, or cultured for 24 h +/ Lactobacillus rhamnosus GG (LGG). Interleukin (IL)-4, IL-10, IL-12, Interferon (IFN)-gamma and total IgE levels were measured in supernatants (ELISA). C. pneumoniae infection led to an increase (>50%) of IgE levels in PBMC from asthmatics, compared with mock-infected on day 10; IgE wasn’t detected in non-asthmatics. C. pneumoniae – infected PBMC from asthmatics increased levels of IL-4 and IFN-gamma after 24 h, compared with PBMC alone; levels of IL-10 and IL-12 were low. When uninfected-PBMC from asthmatics were LGG-stimulated, after 24 h, IL-4 was undetectable, but IL-10, IL-12, and IFN-gamma increased, compared with PBMC alone. Thus, C. pneumoniae infection has the ability to induce allergic responses in PBMC of asthmatics, as evidenced by production of Th2 responses and IgE. Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction The importance of viral infections as infectious triggers of asthma exacerbations is well established [1,2]. The relationship between atypical bacterial infection (Chlamydia pneumoniae, Mycoplasma pneumoniae) and pathogenesis of asthma is a controversial topic [3]; studies investigating this association have been weak or provide conflicting data [4]. This may be due, in part, to inaccurate Abbreviations: PBMC, peripheral blood mononuclear cells; Abs, antibodies; Ig, Immunoglobulin; Th, T helper; TLR, toll like receptor; LGG, Lactobacillus rhamnosus GG; IL, Interleukin; AD, atopic dermatitis; MIF, microimmunofluorescence; MOI, multiplicity of infection; IFN, interferon; TNF, tumor necrosis factor; ELISA, enzyme linked immunosorbent assay; EB, elementary bodies; p.i., post-infection; TLR, tolllike receptor. ⇑ Corresponding author at: SUNY Downstate Medical Center, Dept of Pediatrics, Box 49, 450 Clarkson Ave., Brooklyn, NY 11203, United States. E-mail address: [email protected] (T.A. Smith-Norowitz).
diagnosis of infection [4], insensitive laboratory techniques [4], or poorly designed studies [4]. C. pneumoniae, an obligate intracellular bacterium [5], causes respiratory infection in adults and children and has been implicated in exacerbations of asthma [4]. Studies have shown that C. pneumoniae is capable of causing prolonged respiratory infections in asthmatic and non-asthmatics individuals [6–8]. Previous studies in our laboratory detected increased prevalence of anti-C. pneumoniae IgE Abs in children with wheezing compared with healthy controls and children with pneumonia who were not wheezing [9]. In addition, our laboratory demonstrated that doxycycline suppresses C. pneumoniae-mediated increases in ongoing IgE and IL-4 responses by peripheral blood mononuclear cells (PBMC) of patients with allergic asthma [10]. C. pneumoniae infection activates immune cells to produce cytokines that may contribute to the pathology seen in allergic
http://dx.doi.org/10.1016/j.humimm.2016.02.010 0198-8859/Ó 2016 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
Please cite this article in press as: T.A. Smith-Norowitz et al., Chlamydia pneumoniae enhances the Th2 profile of stimulated peripheral blood mononuclear cells from asthmatic patients, Hum. Immunol. (2016), http://dx.doi.org/10.1016/j.humimm.2016.02.010
2
T.A. Smith-Norowitz et al. / Human Immunology xxx (2016) xxx–xxx
asthma [4]. In addition, infection with C. pneumoniae triggers the production of pathogen-specific IgE in children with chronic respiratory disease, which may contribute to inflammation [11]. In the current study, we examined whether the presence of C. pneumoniae alters IgE levels and/or cytokine profiles in C. pneumoniae- infected PBMC from patients with asthma compared with non-asthmatic controls, using an in vitro model of C. pneumoniae infection and stimulation with Lactobacillus rhamnosus GG (LGG). LGG has been shown to modulate innate immune gene expression and attenuate inflammatory responses to pathogenic bacteria [12]. 2. Methods 2.1. Study participants Adult patients with allergic asthma (N = 11) and healthy nonasthmatic controls (N = 12) (male and female, 18–65 years old) were recruited from the outpatient department at SUNY Downstate Medical Center (Brooklyn, NY); written informed consent and assent was obtained from participants for use of their blood samples for an experimental study. Specific inclusion criteria for enrolled patients included a physician’s diagnosis of stable asthma or current clinically defined persistent asthma symptoms [13], or both, with elevated serum IgE levels (>100 IU/mL). Exclusion criteria included history of chronic immunosuppressive or autoimmune disease, human immunodeficiency virus infection, cancer, recent use of systemic corticosteroids (
