Chlamydia pneumoniae enhances the Th2 profile of stimulated peripheral blood mononuclear cells from asthmatic patients

Human Immunology xxx (2016) xxx–xxx

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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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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 (<30 days) or immunotherapy, tobacco use within the past year, and incomplete follow-up. Non-asthmatic control subjects were defined by absence of asthma, based on clinical criteria [14], with normal serum IgE levels (<100 IU/mL). Peripheral blood (10 mL) was collected at the time of diagnosis, follow-up, or referral to our clinic. All study activities were approved by the SUNY Downstate Medical Center Institutional Review Board (Brooklyn, NY) (study number 08-027), and the procedures were followed were in accordance with institutional guidelines involving human subjects. 2.2. Immunoglobulin determination: total serum IgE Blood was collected and total serum IgE levels were determined in serum using the UniCap Total IgE fluoroenzyme immunoassay (Pharmacia and Upjohn Diagnostics, Freiburg, Germany) performed according to the manufacturer’s recommendations (reference range for healthy adult: 20–100 IU/mL). All tests were performed in the Clinical Diagnostic Laboratory at SUNY Downstate Medical Center (Brooklyn, NY). 2.3. Detection of C. pneumoniae-specific IgG antibodies C. pneumoniae-specific IgG antibodies were measured using the microfluorescence (MIF) test, as previously described [15]. 2.4. Preparation of C. pneumoniae C. pneumoniae TW-183 (ATCC 53592; Manassas, VA) was propagated as previously described [15]. Briefly, HEp-2 cell (ATCC LCL23) monolayers were inoculated with C. pneumoniae and grown to high titers by serial passage in HEp-2 cells. C. pneumoniae elementary bodies (EB) were purified by Urografin (Schering, Berlin, Germany) density gradient centrifugation and were resuspended in sucrose phosphate glutamate buffer (comprising 74.62 g/l sucrose (Sigma, St Louis, MO), 0.517 g/l KH2PO4 (Sigma), 1.643 g/l K2HPO4 (Sigma), and 0.907 g/l potassium glutamate (Sigma)). Titers were determined by infecting HEp-2 cells with serial dilutions of EB suspension aliquots, fixing cells at 72 h post infection

(p.i.), staining with fluorescein-conjugated murine monoclonal genus-specific anti-lipopolysaccharide monoclonal antibody (Pathfinder, Bio-Rad, Hercules, CA), and counting inclusions per well. Aliquots were frozen at 80 °C until use. 2.5. Cell cultures PBMC were separated from blood on a Ficoll-Paque (GE Healthcare, Sweden) gradient (density 1.077). The PBMC were carefully removed using a transfer pipette (VWR Scientific, San Francisco, CA). Cells were washed twice in RPMI-1640 medium (Life Technologies/GIBCO, Grand Island, NY) with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA), and resuspended in complete RPMI 1640 (c-RPMI). c-RPMI contained RPMI-1640 Medium HEPES Modification (Sigma) supplemented with 5 mM L-glutamine (Sigma) and 10% FBS (Atlanta Biologicals). Cells were counted on a hemocytometer (Fisher Scientific, Springfield, NJ), and cell viability was evaluated, as judged by trypan blue (Fisher Scientific) exclusion. For each experimental condition PBMC (1.5  106/mL) were cultured in duplicate in a 24-well flat bottom plate (1 mL/well) (CORNING; Corning, NY) at 37 °C in cRPMI medium in a humidified 5% CO2 atmosphere for up to 12 days. Cell viability was determined at 0, 48 and 240 h (>98%, 95%, and 90%, respectively). Following a 2 h incubation to allow adherence, PBMC cultures were infected with C. pneumoniae or MI and/or stimulated in the presence or absence of LGG (10 billion cells/serving) (Culturelle Digestive Health Probiotic; Culturelle, Cromwell, CT) for up to 12 days. LGG (Culturelle) was serially diluted (1:1, 1:2, 1:4, 1:10) to determine optimal dose. Optimal dose was established in pilot experiments and determined to be a ratio of 1:1 LGG bacteria/cells, for the purpose of cytokine production. It should be mentioned, that in some experiments, PBMC were allowed to adhere to the cell culture plates (2 h), then non-adherent cells were removed and cultured separately from adherent cells. Cytokine assays (IFN-gamma, IL-4) were run using supernatants from each separate cell population listed above. T cell cytokines reported in this study were found in the non-adherent cell population as well as in the unfractionated PBMC cultures but not in the adherent cell population. 2.6. In vitro infection with C. pneumoniae and treatment with LGG PBMC were infected with C. pneumoniae by adding purified EB for 1 h, or treated with a physiologic concentrations of LGG (1:1) (Culturelle), for up to 12 days p.i. at 37 °C in cRPMI in a humidified 5% CO2 atmosphere. The multiplicity of infection (MOI; 0.1) and time points (24 h p.i. for cytokines and 10d p.i. for IgE) used for analysis were selected by kinetic and dose response studies (using MOI of 0.01–10) for optimization of the assay, which revealed peak concentrations and clear distinctive profiles for the respective outcome variables at these time points. Adherent cells were stained with a fluorescein-conjugated murine monoclonal genus-specific anti-lipopolysaccharide antibody (Becton–Dickinson (BD) Biosciences, San Jose, CA) to confirm and quantify infection with C. pneumoniae at 72 h p.i. Two types of controls were used in infection experiments: identical volumes of heat-inactivated purified C. pneumoniae (‘‘mock-infection”) [10] and identical volumes of HEp-2 cell cultures not containing any bacteria processed the same way as the purified C. pneumoniae [16], and lactobacillus (1:1) based on dose–response experiments. 2.7. Cytokine (IL-4, IL-10, IL-12, IFN-gamma) 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-10:

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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BioLegend ELISA MAX Standard; Bio Legend, San Diego, CA) (IL-12, IFN-gamma: Abcam, Cambridge, MA) or IgE ELISA test kits (Bio Quant, San Diego, CA), according to the manufacturer’s recommended procedure. Specifically, cell culture supernatants were collected at either 24 p.i. (cytokines) or 10 days p.i. (IgE), by centrifugation, and samples were stored at 80° until analysis. Supernatants collected for IgE measurement were frozen and thawed three times to release intracellular IgE before centrifugation. All specimens were analyzed in duplicate and standard curves determined. The IgE ELISA was modified for in vitro use by using a low-range standard curve; the sensitivity of the assay was determined by using 2 standard deviations above the mean of 10 negative control measurements (0.3 ng/mL). Plates were read using an automated microplate reader (Model ELx800; Bio-Tek Instruments, Winooski, VT), with a 450-nm measurement filter. 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 assays were: IL-4: 3.9 pg/mL; IL-10: 3.9 pg/mL; IL-12: <0.75 pg/mL; IFN-gamma: <5.0 pg/mL. 2.8. Quantitative real time polymerase chain reaction (qPCR) Extraction of DNA from nasopharyngeal swabs and PBMC was performed according to manufacturer’s recommendations (Qiagen Inc., Valencia, Calif.). For PBMC cultures supernatants with adherent and non-adherent cells were collected and DNA extracted. Detection and quantification of C. pneumoniae and M. pneumoniae DNA was performed according to Apfalter et al. [17] and Waring et al. [18] respectively, using TAQMan technology based quantitative real-time PCR on the Light Cycler 2.0 platform (software version 4.0, Roche). Three replicates of each dilution of the standards and sample extracts were tested; an additional replicate was checked for PCR inhibitors and inhibited specimens were retested 1:10 diluted. qPCR analyses were considered negative, if the crossing point (Cp) values exceeded 45 cycles. 2.9. Statistical analysis Data are expressed as means with standard deviation (SD) unless otherwise indicated. Students T test and the nonparametric Wilcoxon Signed Ranks Test were used to compare differences in means of normally and non-normally distributed data, respectively. The Pearson Correlation Test was used to assess the degree of correlation for continuous variables. A 2-sided P value of <0.05 was taken to indicate statistical significance for all comparisons. All data and statistical analyses were performed using SPSS for Windows, version 12.0 software (Chicago, IL). 3. Results 3.1. Participant characteristics and demographics We enrolled11 patients with allergic asthma (64% female, 36% male; 46 ± 17 years) and 12 healthy non-asthmatic controls (58% female, 42% male; 36 ± 12 years). All asthmatic subjects were serum IgE positive (614 ± 512 IU/mL). None of the healthy subjects had a history of asthma or allergic rhinoconjunctivitis; serum IgE levels were lower in controls (<100 IU/mL). All patients were classified as having moderate persistent asthma and were treated with inhaled corticosteroids. 3.2. Serological and nucleic amplification testing for C. pneumoniae Asthmatic patients (55%) had C. pneumoniae specific-IgG microfluorescence (MIF) titers >1:16 with a median titer of 1:32.

Control subjects (50%) had C. pneumoniae IgG MIF titers >1:16, with a median titer of 1:32. All subjects (asthmatic and non-asthmatics) tested negative for C. pneumoniae and M. pneumoniae, as determined by qPCR (nasopharyngeal swabs). 3.3. Distributions of blood lymphocyte subpopulations pre- and postchlamydia infection PBMC obtained from asthmatic and non-asthmatic subjects had similar numbers of CD3+CD4+ (45% ± 3.0, 48% ± 4.0), CD3+CD8+ (21 + 3.0, 28 + 8.0) T cells, and CD19+ B cells (4.0 ± 1.0, 6.0 ± 1.0) pre-chlamydia infection (Table 1). In asthmatic subjects, after 24 h mock-infection and 24 h post chlamydia-infection numbers of CD3+CD4+ T cells slightly increased, numbers of CD3+CD8+ T cells decreased by 40% and 50%, respectively, numbers of CD19 + B cells remained the same. In non-asthmatics, after 24 h mockinfection and 24 h post chlamydia-infection numbers of CD3+CD4 + T cells slightly increased, numbers of CD3+CD8+ T cells slightly increased, and numbers of CD19+ B cells decreased (50%) (Table 1). 3.4. Cytokines (IFN-gamma, IL-4) levels in adherent and non-adherent cells in response to C. pneumoniae When PBMC from asthmatic patients were cultured with C. pneumoniae (MOI = 0.1), levels of IFN-gamma were low (0 pg/ mL) in adherent cells, were high (49.9 pg/mL) in non-adherent cells, and higher in unfractionated cells (96 pg/mL). Levels of IL-4 were low in adherent cells (1.6 pg/mL), and increased slightly in non-adherent cells (2.5 pg/mL) and unfractionated cells (3.2 pg/ mL) (Fig. 1). 3.5. Effect of C. pneumoniae infection on IgE responses induced in vitro When PBMC from asthmatic patients were cultured with C. pneumoniae, increased mean levels (>50%) of IgE were detected in supernatants on day 10, compared with mock-infected PBMC (P < 0.03) (Fig. 2). In contrast, when PBMC from non-asthmatic subjects were cultured with C. pneumoniae or mock-infected, mean levels of IgE were undetected in supernatants on day 10 (<0.3 ng/ mL) (Data not shown). In vitro IgE responses were higher in asthmatic patients with IgG anti-C. pneumoniae MIF titers P1:16 compared with asthmatic patients with IgG anti- C. pneumoniae MIF titers <1:16 and control subjects with C. pneumoniae MIF titers P1:16 or <1:16 (Fig. 3).

Table 1 Distributions of lymphocyte subpopulations in peripheral blood mononuclear cells (PBMC) of asthmatic and non-asthmatic human subjects pre and post chlamydia infection. Subject/time

CD3+CD4+ %

CD3+CD8+ %

CD19+ %

Asthma Pre 24 h mock 24 h p.i.

45 ± 3.0 50 ± 7.0 54 ± 8.0

21 ± 3.0 13 ± 7.0 11 ± 5.0

4.0 ± 1.0 nt 4.0 + 2.0

Non-asthma Pre 24 h mock 24 h p.i.

48 ± 4.0 50 + 3.0 51 ± 4.0

28 ± 8.0 29 ± 8.0 30 ± 7.0

6.0 ± 1.0 4.0 ± 1.0 3.0 ± 1.0

* The distributions of lymphocyte subpopulations (CD3+CD4+, CD3+CD8+, CD19+) in peripheral blood mononuclear cells (PBMC) obtained from asthmatic (N = 3) and non-asthmatic (N = 3) human subjects pre-chlamydia infection, 24 h mock infection (identical volumes of heat-inactivated purified C. pneumoniae), and 24 h post infection (MOI = 0.1) (representative experiments). Data are expressed as mean percentage (%) of positive cells ± standard deviation (SD).

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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IFN - γ

50

0 Adherent

Non-Adherent

Unfraconated

IgE (ng/ml)

pg/mL

100

Cells

IL-4 10

pg/mL

8

> 1:16

6

< 1:16

> 1:16

asthma

4

< 1:16 control

Cpn MIF ter 2 0 Adherent

Non-Adherent

Unfraconated

Cells Fig. 1. Cytokines levels in adherent and non-adherent cells in response to Chlamydia. Cytokine (IFN-gamma, IL-4) levels (concentration in culture with Chlamydia minus concentration in culture without Chlamydia) were measured in culture after 24 h in adherent cells, non-adherent cells and unfractionated cells. Cells were fractionated by allowing PBMC to adhere for 2 h before separating them into cell culture chambers, followed by infection with Chlamydia (MOI = 0.1). Supernatants were collected after 24 h P.I. and assayed for cytokines (ELISA). Data are from a representative experiment with blood obtained from one of two different asthma donors, with similar results. Data are expressed as pg/mL.

Fig. 3. Effect of C. pneumoniae infection on production of IgE in vitro in relationship to C. pneumoniae microfluorescence (MIF) titers. IgE responses were higher in asthmatic patients with IgG anti C. pneumoniae MIF titers P1:16 (N = 6) compared with asthmatic patients with IgG anti-C. pneumoniae MIF titers <1:16 (N = 4) and non-asthmatic control subjects with C. pneumoniae MIF tiers P1:16 or <1:16 (N = 12). Data are expressed as ng/mL. IgE response was defined as IgE in infected PBMC minus IgE in uninfected PBMC. Cpn: C. pneumoniae.

were detected in supernatants after 24 h, compared with PBMC alone (Fig. 4A, D); levels of IL-10 and IL-12 in supernatants were low (P > 0.05) (Fig. 4B, C). 3.7. Effect of LGG stimulation on cytokine responses induced in vitro When PBMC from asthmatic patients were cultured with uninfected PBMC and stimulated with LGG, levels of IL-4 were undetectable in both uninfected PBMC and PBMC stimulated with LGG after 24 h (Fig. 4A). However, levels of IL-10 and IFN-gamma increased in supernatants of PBMC stimulated with LGG compared with PBMC alone after 24 h (Fig. 4B, D) (P = 0.003, P = 0.043, respectively). There was a trend to increased levels of IL-12 in PBMC stimulated with LGG, compared with PBMC alone, which was not statistically significant (P > 0.05) (Fig. 4C). 4. Discussion

Fig. 2. Effect of C. pneumoniae infection on production of IgE in vitro. Human PBMC (1.5  106/mL) were obtained from asthmatic subjects (N = 10) (see Section 2) and cultured in the absence (mock-infected) or presence of C. pneumoniae (MOI = 0.1) for 10 days, and levels of IgE were measured in supernatants. IgE levels were significantly higher in supernatants from C. pneumoniae-infected compared with mock-infected PBMC (P < 0.03). Data are expressed as ng/mL.

3.6. Effect of C. pneumoniae infection on cytokine responses induced in vitro When PBMC from asthmatic patients were cultured with C. pneumoniae, increased levels of IL-4 (P = 0.043) and IFN-gamma

This study examined T helper responses in C. pneumoniaeinfected PBMC in patients with asthma compared to nonasthmatic control subjects. The results demonstrated that C. pneumoniae infection can induce allergic responses in PBMC of asthmatics, as evidenced by increased production of Th2type cytokines (IL-4) and induction of IgE responses. In contrast, increased Th1 responses elicited by LGG in vitro in PBMC from asthmatic patients may result in part, from morphologic properties, including the presence of a bacterial cell wall peptidoglycan, which can suppress IgE production [10]. While recent studies have described an intact peptidoglycan polymer surrounding chlamydial cells [16], the effects on host cell responses differ from LGG, indicating some key differences in the function of the peptidoglycan, and the ability to stimulate immune cells of the peptidoglycan. In contrast to the majority of bacteria, Chlamydia has a very small amount of polypeptide wall, which is expressed only during cell septation [19].

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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Fig. 4. Effect of C. pneumoniae infection on cytokine (IL-4, IL-10, IL-12, IFNgamma) responses in vitro. Human PBMC (1.5  106/mL) were obtained from asthmatic subjects (N = 11) and cultured with either C. pneumoniae (MOI = 0.1) or Lactobacillus GG (1:1 ratio of bacteria to PBMC) or alone for 24 h, and levels of cytokines (Panel A: IL-4, Panel B: IL-10, Panel C: IL-12, Panel D: IFN-gamma) were measured in supernatants. Data are expressed as pg/mL (mean ± standard deviation). Cytokine response was defined as cytokine of interest in infected PBMC minus cytokine of interest in uninfected PBMC.

C. pneumoniae infection led to a significant increase (>50%) of IgE levels in supernatants of PBMC from asthmatic patients, compared with mock-infected PBMC on day 10 p.i. However, IgE was not detected in supernatants from either C. pneumoniae infected or mock-infected PBMC from non-asthmatics. This would suggest that C. pneumoniae infection may promote allergic inflammation and the ongoing production of IgE. In addition, both asthmatic patients and non-asthmatic controls had similar seroprevalence rates and titers of C. pneumoniae IgG, indicating that both groups might have had similar rates of past infection with C. pneumoniae. Thus, the difference in IgE responses between the two groups cannot be attributed to differences in exposure alone. These findings are similar and in agreement with our past studies that reported that when PBMC from adult patients with allergic asthma were cultured in the presence of C. pneumoniae (MOI = 0.1), increased production of IgE production was observed on day 10, compared with uninfected PBMC (P = 0.008) [10]. Similarly, Mukouyama et al. reported a spontaneous production of IgE by PBMC from children with asthma; levels of IgE in culture supernatants ranged from 0.1 to 15.0 IU/mL [20]. Past studies in our laboratory also observed spontaneous IgE production, along with a significant increase in levels of IL-4, and a correlation between levels of C. pneumoniae-induced IgE and IL-4, suggesting that C. pneumoniae infection mediates IgE increases through Th2 lymphocyte activation [10]. Prior literature has established that following C. pneumoniae infection, ongoing inflammation may contribute to the severity and progression of asthma [8]. In the current study, we also observed that C. pneumoniae-infected PBMC from asthmatic patients significantly increased levels of IL-4 and IFN-gamma, while levels of IL-10 and IL-12 were low. However, when uninfected PBMC from asthmatic patients were stimulated with LGG, levels of IL-4 were undetectable; levels of IL-10 and IFN-gamma increased significantly, and there was a trend to increased levels of IL-12, which were not statistically significant. Thus, it could be that the Th2 bias observed in C. pneumoniae- infected PBMC from asthmatics may be due to differential stimulation and/or activation of receptors for pathogen-associated molecular patterns (PAMP). Toll-like receptors (TLR) are necessary for host defense against

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microbes in that they recognize PAMP [21]. In vitro studies have demonstrated that TLR2 and TLR4 are involved in recognition of C. pneumoniae [22]; in vivo studies showed that TLR2 is involved in the recognition of C. pneumoniae, as demonstrated by impaired cytokine and chemokine secretion [23]. Thus, the present study is in agreement with earlier studies in our laboratory that reported a significant increase in the secretion of IL-4, a critical cytokine for the induction of production of IgE responses, and low levels of IFNgamma from C. pneumoniae-infected PBMC obtained from allergic asthmatics compared with controls [10]; the results suggest that C. pneumoniae infection may promote allergic inflammation (Th2 responses), as well as the ongoing production of IgE responses [10]. Antigen specific T cells play a critical role in the immune control of viral and bacterial pathogens [24]; however, C. pneumoniae is a pathogen known for persistent infections [5]. Bunk et al., demonstrated the presence of circulating memory CD4+ T cells in PBMC of healthy human blood donors that when stimulated with C. pneumoniae, produced IL-2 and IFN-gamma, which may reflect the efficient immune control of viral infection [24]. Other cells involved in C. pneumoniae infection are dendritic cells, which lead to dissemination of infection from the lungs [25]. Flego et al. demonstrated that C. pneumoniae modulates human monocyte-derived dendritic cell function which produced IL-12p70, IL-1Beta, IL-6, and IL-10, and drove a mixed Type1/Type 17 pro-inflammatory response [25]; the intracellular pathways triggered by C. pneumoniae involved TLR2 [25]. However, when ERK1/2 inhibitor was included, IL-12p70 and IL-10 release was reduced and T cell polarization shifted toward a Th2 profile [25]. Thus, TLR and ERK1/2 induced by C. pneumoniae may affect dendritic cell function that contributes to a Th1 pro-inflammatory response [25]. In the current study, when uninfected PBMC were stimulated with LGG, a Th1 dominance was observed, perhaps through stimulating the production of IFN-gamma (pro-inflammatory cytokine) and inhibition of IL-4 and IL-10 (anti-inflammatory cytokines), which can differentiate the immune response to a Th2 bias. These observations are consistent with previous studies that reported LGG has in vitro effects on enhanced IL-10 and IFN-gamma release of mononuclear cells [26]. In contrast, Lin et al. demonstrated that other probiotic strains, Lactobacillus paracasei BB5 and/or Lactobacillus rhamnosus BB1, can induce apoptosis of Th2 lymphocytes (CD4+ IL-4+ T cells) and inhibit airway hyper-responsiveness and inflammation in a mouse model [27]. However, apoptosis of Th2 lymphocytes was not studied in the current study. LGG has been shown to diminish mast cell allergy-related activation by down-regulation of the expression of high-affinity IgE and histamine receptor genes, perhaps by inducing a proinflammatory response [29]. It has also been reported that mast cells stimulated with LGG, L rhamnosus Lc705 (Lc705), Bifidobacterium animalis ssp. lactis Bb12 (Bb12), a combination of both, or with C. pneumoniae also induced expression of a gene that encodes IL-10 [28]. However, mast cells stimulated with C. pneumoniae up-regulated only IL-10 without affecting the genes FCER1 (high affinity IgE receptor) and HRH4 (histamine H4 receptor) [28]. Thus, stimulation of mast cells with C. pneumoniae may not have the same clinical effect as stimulation with specific probiotic bacteria [28]. The infection life cycle of C. pneumoniae is characterized by two developmental stages: the elementary bodies, which are infectious but have reduced metabolic activity [29], and the replicative reticulate bodies that are not infectious [30–33]. C. pneumoniae has an atypical cell division mechanism, which is independent of an FtsZ (tubulin) homologue, and is not well understood [34]. In rod-shaped bacteria, including Lactobacillus species, the FtsZ protein tethers incorporation of cell wall building blocks at the developing septum [35]. Recent literature has reported evidences of peptidoglycan-like material in both Chlamydiaceae and

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Chlamydia-related bacteria [34]. In addition, peptidoglycan synthesis is required for localization of the newly described septal proteins RodZ and N1pD21 [34]. Thus, it could be, the presence of a small amount of peptidoglycan material found in C. pneumoniae can affect the cytokine profile during infection It has been reported that cell-mediated immunity responses (CD4+ T cells) play a critical role in protective immunity to Chlamydia infection [36]. However, recent evidence supports a prominent role for B cell-mediated immunity in some intracellular infection models, including Chlamydia [36]. However, there exists concern regarding the ability to clear chlamydial infections in the presence of a Th2-biased T cell response and the development of persistent infection [10]. Suppression of IFN-gamma responses may inhibit clearance of C. pneumoniae through cell-mediated immunity, specifically Th1 responses, which are crucial in the clearance of infections caused by C. pneumoniae [37]. Consistent with the literature, one of the defining features of chlamydial infections is the ability to cause persistent infection [5]; these persistent infections have been reported in patients with asthma and may be associated with lack of strong Th1 responses [10,38]. Further, prior literature has shown an association between atypical bacterial infection (C. pneumoniae, M. pneumoniae) and chronic stable asthma and exacerbations of asthma [4]; however, this does not specify a causative role for infection in asthma pathogenesis, but may indicate higher susceptibility to infection and/or possible increased frequency of detection [4]. Some study limitations to our findings should be noted including: (1) small sample size, (2) serologic methods for confirmation of timing of infection may be difficult, and thus incidence of infection is not known, and (3) the true effect of C. pneumoniae-infection may be difficult to show in subjects already up-regulated toward a Th2 pattern, as in asthmatic subjects. In addition, an optimal immune response by T cells may require a longer period of incubation, beyond 24 h. We were only able to stimulate our cells with LGG for 24 h because after 24 h LGG becomes toxic to the cells and results in apoptosis. In summary, our finding and those of previous studies [10] support consideration that C. pneumoniae infection can modulate both Th2 and IgE responses in PBMC from asthmatic patients. These findings suggest that C. pneumoniae may have the ability to induce allergic responses in PBMC, through mechanisms to be determined. However, these data represent only preliminary evidence of a relationship between C. pneumoniae infection and allergic response in patients with asthma. Future studies should examine underlying genetic predispositions to abnormal inflammatory responses to C. pneumoniae and identify immunologic and epigenetic pathways involved in modulating inflammatory/IgE responses through identification of biomarkers involved in these purported associations. Thus, this study may provide a basis for development of a novel therapeutic method for prevention of allergic responses. Disclosure The authors declare no competing financial interest to disclose. Conflict of interest The authors declare no conflict of interest to disclose. Acknowledgements We thank Kevin Norowitz, M.D. (Department of Pediatrics, SUNY Downstate Medical Center) for editing and critical reading of the manuscript. This study was funded by a NY State Divisional Grant.

References [1] S.L. Johnston, P.K. Pattermore, G. Sanderson, S. Smith, F. Lampe, L. Josephs, P. Symington, S. O’Toole, S.H. Myint, D.A. Tyrrell, et al., Community study of role of viral infections in exacerbations of asthma in 9–11 year old children, BMJ 310 (1995) 1225–1229. [2] S.L. Johnston, P.K. Pattemore, G. Sanderson, S. Smith, M.J. Campbell, L.K. Josephs, A. Cunningham, B.S. Robinson, S.H. Myint, M.E. Ward, et al., The relationship between upper and respiratory infections and hospital admissions for asthma: a time-trend analysis, Am. J. Respir. Crit. Care Med. 154 (1996) 654–660. [3] R.F. Lemanske Jr., Is asthma an infectious disease?, Chest 123 (2003) 385S– 390S [4] S.L. Johnston, R.J. Martin, Chlamydophila pneumoniae and Mycoplasma pneumoniae a role in asthma pathogenesis?, Am J. Respir. Crit. Care Med. 172 (2005) 1078–1089. [5] M.R. Hammerschlag, S.A. Kohlhoff, C.A. Gaydos, Chlamydia pneumoniae, in: G. L. Mandell, J.E. Bennett, R. Dolin (Eds.), Principles and Practice of Infectious Diseases, 8th ed., Elsevier, Inc., Philadelphia, PA, 2014, pp. 2174–2182. [6] U. Emre, P.M. Roblin, M. Gelling, et al., The association of Chlamydia pneumoniae infection and reactive airway disease in children, Arch. Pediatr. Adolesc. Med. 148 (1994) 727–731. [7] M.R. Hammerschlag, K. Chirgwin, P.M. Roblin, et al., Persistent infection with Chlamydia pneumoniae following acute respiratory illness, Clin. Infect. Dis. 14 (1992) 178–182. [8] R.J. Martin, M. Kraft, H.W. Chu, et al., A link between chronic asthma and chronic infection, J. Allergy Clin. Immunol. 107 (2001) 595–601. [9] U. Emre, N. Sokolovskaya, P.M. Roblin, et al., Detection of anti-Chlamydia pneumoniae IgE in children with reactive airway disease, J. Infect. Dis. 172 (1995) 265–267. [10] M.S. Dzhindzhikhashvili, R. Joks, T.A. Smith-Norowitz, H.G. Durkin, K. Chotikanatis, E. Estrella, M.R. Hammerschlag, S.A. Kohlhoff, Doxycycline suppresses Chlamydia pneumoniae-mediated increases in ongoing immunoglobulin E and interleukin-4 responses by peripheral blood mononuclear cells of patients with allergic asthma, J. Antimicrob. Chemother. 68 (2013) 2363–2368. [11] S. Ikezawa, Prevalence of Chlamydia pneumoniae in acute respiratory tract infection and detection of anti-Chlamydia pneumoniae-specific IgE in Japanese children with reactive airway disease, Kurume Med. J. 48 (2) (2001) 165–170. [12] K. Ganguli, M.C. Collado, J. Rautava, L. Lu, R. Satokari, I. von Ossowski, J. Reunanen, W.M. de Vos, A. Palva, E. Isolauri, S. Salminen, W.A. Walker, S. Rautava, Lactobacillus rhamnosus GG and its Spa C pilus adhesion modulate inflammatory responsiveness an TLR-related gene expression in the fetal human gut, Pediatr. Res. 77 (2014) 528–535. [13] M.K. Cowen, D.B. Wakefield, M.M. Cloutier, Classifying asthma severity: objective versus subjective measures, J. Asthma 44 (2007) 711–715. [14] EPR-3. Expert Panel Report 3: Guidelines for the Diagnosis and Management of Asthma (EPR-3 2007), U.S. Department of Health and Human Services; National Institutes of Health; National Heart, Lung, and Blood Institute; National Asthma Education and Prevention Program, 2007, pp. 40–43. . [15] S.P. Wang, J.T. Grayston, Microimmunofluorescence serological studies with the TWAR organism, in: D. Oriel, G. Ridgeway (Eds.), Chlamydial Infections: Proceedings of the Sixth International Symposium on Human Chlamydial Infections, Cambridge University Press, Cambridge, 1986, pp. 329–332 (I think there is a better reference, use our Mandell chapter). [16] P.M. Roblin, W. Dumornay, M.R. Hammerschlag, Use of HEp-2 cells for improved isolation and passage of Chlamydia pneumoniae, J. Clin. Microbiol. 1992 (30) (1992) 1968–1971. [17] P. Apfalter, W. Barousch, M. Nehr, et al., Comparison of a new quantitative ompA-based real-time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays, J. Clin. Microbiol. 41 (2003) 592–600. [18] A.L. Waring, T.A. Halse, C.K. Csiza, et al., Development of a genomics-based PCR assay for detection of Mycoplasma pneumoniae in a large outbreak in New York State, J. Clin. Microbiol. 39 (2001) 1385–1390. [19] S. Pilhofer, J. Lutkenhaus, Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA, Mol. Microbiol. 55 (2005) 1722–1734. [20] T. Mukouyama, W. Ita, K. Ichikawa, et al., Spontaneous IgE production in asthmatic children, Arerugi 43 (1994) 474–481. [21] R. Medzhitov, Toll-like receptors and innate immunity, Nat. Rev. Immunol. 1 (2001) 135–145. [22] M.G. Netea, B.J. Kullberg, J.M. Galama, A.F. Stalenhoef, C.A. Dinarello, J.W. van der Meer, Non-LPS components of Chlamydia pneumoniae stimulate cytokine production through Toll-like receptor 2-dependent pathways, Eur. J. Immunol. 32 (2002) 1188–1195. [23] N. Rodriguez, N. Wantia, F. Fend, S. Durr, H. Wagner, T. Miethke, Differential involvement of TLR2 and TLR4 in host survival during pulmonary infection with Chlamydia pneumoniae, Eur. J. Immunol. 36 (2006) 1145–1155. [24] S. Bunk, H. Schaffert, B. Schmid, C. Goletz, S. Zeller, M. Borisova, F. Kern, J. Rupp, C. Hermann, Chlamydia pneumoniae-induced memory CD4+ T-cell activation in human peripheral blood correlates with distinct antibody response patterns, Clin. Vaccine Immunol. 17 (2010) 705–712.

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

T.A. Smith-Norowitz et al. / Human Immunology xxx (2016) xxx–xxx [25] D. Flego, M. Bianco, A. Quattrini, F. Mancini, M. Carollo, I. Schiavoni, A. Ciervo, C.M. Ausiello, G. Fedele, Chlamydia pneumoniae modulates human monocytederived dendritic cells functions driving the induction of a Type 1/Type 17 inflammatory response, Microbes Infect. 15 (2) (2013) 105–114. [26] M.V. Kopp, M. Goldstein, A. Dietschek, J. Sofke, A. Heinzmann, R. Urbanek, Lactobacillus GG has in vitro effects on enhanced interleukin-10 and interferon-gamma release of mononuclear cells but no in vivo effects in supplemented mothers and their neonates, Clin. Exp. Allergy 38 (2008) 602– 610. [27] W.H. Lin, C.R. Wu, H.Z. Lee, Y.H. Kuo, H.S. Wen, T.Y. Lin, C.Y. Lee, S.Y. Huang, C. Y. Lin, Induced apoptosis of Th2 lymphocytes and inhibition of airway hyperresponsiveness and inflammation by combined lactic acid bacteria treatment, Int. Immunopharmacol. 15 (4) (2013) 703–711. [28] A. Oksaharju, M. Kankainen, R.A. Kekkonen, K.A. Kindstedt, P.T. Kovanen, R. Korpela, M. Miettinen, Probiotic Lactobacillus rhamnosus downregulates FCER1 and HRH4 expression in human mast cells, World J. Gastroenterol. 17 (2011) 750–759. [29] G. Greub, The medical importance of Chlamydiae, Clin. Microbiol. Infect. 15 (2009) 2–3. [30] B.S. Sixt, A. Siegl, C. Muller, et al., Metabolic features of Protochlamydia amoebophilia elementary bodies-a link between activity and infectivity in Chlamydiae, PLoS Pathog. 9 (2013) e1003553.

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[31] R.R. Friis, Interaction of L cells and Chlamydia psittaci: entry of the parasite and host responses to its development, J. Bacteriol. 110 (1972) 706–721. [32] G. Greub, D. Raoult, Crescent bodies of Parachlamydia acanthamoeba and its life cycle within Acanthamoeba polyphaga: an electron micrograph study, Appl. Environ. Microb. 68 (2002) 3076–3084. [33] Y.M. Abdelrahman, R.J. Belland, The chlamydial developmental cycle, FEMS Microbiol. Rev. 29 (2005) 949–959. [34] N. Jaaquier, P.H. Viollier, G. Greub, The role of peptidoglycan in chlamydial cell division: towards resolving the chlamydial anomaly, FEMS Microbiol. Rev. 39 (2015) 262–275. [35] A. Gaballah, A. Kloeckner, C. Otten, H.-G. Sahl, B. Henrichfreise, Functional analysis of the cytoskeleton protein MreB from Chlamydophila pneumoniae, PLoS One 6 (10) (2011) e25129. [36] L.-X. Li, S.J. McSorley, A re-evaluation of the role of B cells in protective immunity to Chlamydia infection, Immunol. Lett. (2015), http://dx.doi.org/ 10.1016/j.imlet.2015.02.004. [37] S. Holme, J. Latvala, R. Karttunen, et al., Cell-mediated immune response during primary Chlamydia pneumoniae infection, Infect. Immun. 68 (2000) 7156–7158. [38] U. Emre, N. Sokolovskaya, P.M. Roblin, J. Schachter, M.R. Hammerschlag, Detection of anti-Chlamydia pneumoniae IgE in children with reactive airway disease, J. Infect. Dis. 172 (1) (1995) 265–267.

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