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. 2013 Sep;81(9):3366–3374. doi: 10.1128/IAI.00334-13

Severity of Allergic Airway Disease Due to House Dust Mite Allergen Is Not Increased after Clinical Recovery of Lung Infection with Chlamydia pneumoniae in Mice

Pavel Dutow a, Sandra Lingner b, Robert Laudeley a, Silke Glage c, Heinz-Gerd Hoymann d, Anna-Maria Dittrich b, Beate Fehlhaber a, Meike Müller d, Armin Braun e, Andreas Klos a,✉
Editor: R P Morrison
PMCID: PMC3754214  PMID: 23817611

Abstract

Chlamydia pneumoniae is associated with chronic inflammatory lung diseases like bronchial asthma and chronic obstructive pulmonary disease. The existence of a causal link between allergic airway disease and C. pneumoniae is controversial. A mouse model was used to address the question of whether preceding C. pneumoniae lung infection and recovery modifies the outcome of experimental allergic asthma after subsequent sensitization with house dust mite (HDM) allergen. After intranasal infection, BALB/c mice suffered from pneumonia characterized by an increased clinical score, reduction of body weight, histopathology, and a bacterial load in the lungs. After 4 weeks, when infection had almost resolved clinically, HDM allergen sensitization was performed for another 4 weeks. Subsequently, mice were subjected to a methacholine hyperresponsiveness test and sacrificed for further analyses. As expected, after 8 weeks, C. pneumoniae-specific antibodies were detectable only in infected mice and the titer was significantly higher in the C. pneumoniae/HDM allergen-treated group than in the C. pneumoniae/NaCl group. Intriguingly, airway hyperresponsiveness and eosinophilia in bronchoalveolar lavage fluid were significantly lower in the C. pneumoniae/HDM allergen-treated group than in the mock/HDM allergen-treated group. We did observe a relationship between experimental asthma and chlamydial infection. Our results demonstrate an influence of sensitization to HDM allergen on the development of a humoral antibacterial response. However, our model demonstrates no increase in the severity of experimental asthma to HDM allergen as a physiological allergen after clinically resolved severe chlamydial lung infection. Our results rather suggest that allergic airway disease and concomitant cellular changes in mice are decreased following C. pneumoniae lung infection in this setting.

INTRODUCTION

Bronchial asthma is a common and complex inflammatory disease of the airways in both adults and children. Immunologically, in the majority of cases, bronchial asthma is characterized by an inflammatory influx dominated by eosinophils but also comprising antigen-presenting cells, T cells, and mast cells whose complex interplay leads to a type II polarized T cell-driven immune response with the hallmark cytokines interleukin-4 (IL-4), IL-5, and IL-13. Histologically, apart from the influx of inflammatory cells, mucus hypersecretion, smooth muscle hypertrophy, and characteristic subepithelial changes that are summarized as “remodelling” constitute the defining features of bronchial asthma. In the subtype of allergic bronchial asthma, specific allergens, such as pollens or house dust mite (HDM) allergen, can be identified as causal agents whose inhalation provokes an exaggerated T helper type II (Th2) polarized immune response with the ensuing features mentioned above and recurrent episodes of airway narrowing leading to cough and dyspnea. Past research has identified a multitude of mediators that create a complex network implicated in the induction, maintenance, and modulation of the clinical and paraclinical characteristics of bronchial asthma (1–5). Among these mediators, the anaphylatoxin C3a, a cleavage product of complement factor C3 that binds to its cognate receptor, can modify airway hyperresponsiveness (AHR) and airway inflammation in asthma (6).

While bronchial asthma is one of the most common chronic diseases of childhood in industrialized countries, its prevalence in developing countries is much lower (7–9), which has led to the formulation of the “hygiene hypothesis,” which postulates that the increased frequency seen in countries with a “westernized lifestyle” is attributed to less contact with microbes. The lack of childhood infections is thought to promote an inappropriate maturation of the immune system with a predominant Th2 phenotype (10–14).

Chlamydia pneumoniae is a Gram-negative, obligate intracellular bacterium that is responsible for respiratory diseases like community-acquired pneumonia, bronchitis, sinusitis, or pharyngitis (15–17). Immunity to C. pneumoniae is characterized by the recruitment of neutrophils, macrophages, and T and B cells and by a strong antibody response accompanied by a T helper cell type 1 (Th1) response and the secretion of IL-1α/β, IL-6, IL-8, IL-12, tumor necrosis factor alpha (TNF-α), granulocyte-macrophage colony-stimulating factor, and gamma interferon (IFN-γ) (15, 18, 19). High seroprevalence with increased C. pneumoniae-specific antibody titers indicates almost ubiquitous contact with this pathogen. Apart from the consequences of acute infection, C. pneumoniae is associated with chronic obstructive pulmonary disease (COPD) (20) and is controversially discussed as being connected to bronchial asthma (15, 21). Since the early 1990s, clinical studies reviewed by Johnston and Martin have implicated infections caused by atypical bacteria like C. pneumoniae and Mycoplasma pneumoniae in the development or exacerbation of both acute and chronic asthma (22). Recent studies support these observations (23, 24). However, others did not find a correlation between C. pneumoniae infection and asthma (25, 26).

Only a few investigations have experimentally addressed the possibility of a causal relationship between chlamydiae and allergic airway disease (AAD) in animal models. Blasi et al. reported in 2007 that C. pneumoniae induces a sustained AAD and inflammation in mice and suggested that this pathogen can worsen and/or provoke breathlessness in patients with asthma and COPD (27). However, no allergen was administered in that study.

Additionally, models applying allergens mostly utilize rather artificial allergens like ovalbumin (28–30) or human serum albumin (HSA) (31, 32). Nevertheless, we have learned from these studies that ongoing low-dose (mild) infection with C. pneumoniae can increase eosinophilic airway inflammation and goblet cell hyperplasia if HSA exposure is started on day 5 postinfection (p.i.), whereas severe infection and prolonged low-dose infection (day 10 p.i.) do not have this effect.

Additionally, in models examining the relationship between respiratory Chlamydia and experimental allergic asthma, the mouse pathogen C. muridarum was also used (28–30, 33). According to these studies, C. muridarum infection at a young age affects hallmarks of AAD in adult mice by enhancing its severity.

On the basis of the controversial discussion of correlation and causality, we investigated the potential link between acute C. pneumoniae lung infection and subsequent experimental asthma induced by HDM allergen as a physiological allergen. Our data indicate that a clinically settled severe chlamydial lung infection does not exacerbate the development of experimental allergic asthma in a mouse model. Yet, as demonstrated by the increased anti-C. pneumoniae antibody levels in C. pneumoniae/HDM allergen-treated mice, an interaction between the two lung diseases occurs. In addition, our results suggest a decreased severity of the AAD (AHR) and reduced concomitant cellular changes following acute C. pneumoniae infection.

MATERIALS AND METHODS

Chlamydial culture.

C. pneumoniae CWL029 (ATCC VR-1310) was propagated in BHK-21 cells as previously described (34). Before being used in our experiments, the bacterial stock was proven to be Mycoplasma free by PCR. Infectivity of the elementary bodies, measured as inclusion-forming units (IFU), was determined by titration in HeLa-T cells as previously described (35). For mock-infected controls, BHK-21 cells were processed identically but without Chlamydia and diluted in phosphate-buffered saline at the same ratio that was used for cells mixed with a bacterial suspension.

Experimental model.

All animal procedures were approved by the local district government and carried out in strict accordance to the German legal guidelines for the protection of animal life (permit 33.9-42502-04-11/0537). For animal experiments, 4-week-old female BALB/c mice were ordered from Charles River (WIGA, Sulzfeld, Germany) and used at the age of 6 weeks for experiments. The experimental setup consisted of seven different groups treated in the following ways: C. pneumoniae/HDM allergen treatment, C. pneumoniae/NaCl treatment, mock/HDM allergen treatment, mock/NaCl treatment, no treatment, C. pneumoniae infection for 5 days, and mock infection for 5 days.

Infection with C. pneumoniae.

Intranasal (i.n.) infection was performed with 1.3 × 107 IFU of C. pneumoniae CWL029 or mock material in 0.9% NaCl in a final volume of 30 μl per mouse. The infection was performed as described previously (36). Mice were monitored daily; body weights and clinical scores were recorded until day 27 p.i. (or day 5 if indicated). In brief, the appearance of mice was assessed by using the following parameters: vocalization, body posture, locomotion, breathing, piloerection, overall attention or curiosity, and secretion (eyes, nose, and anal region). Dysfunction in each parameter was rated as 1 or 2 points, depending on the severity of presentation. The overall body condition of the mice was determined by adding all points, resulting in a clinical score of untroubled (0 points) to moribund (≥10 points; in this case, mice were euthanized) (35, 37).

Sensitization with HDM extract.

From day 28 p.i. on, mice were sensitized to HDM allergen (HDM extract; Greer Inc.) by i.n. administration of 25 μg protein content of HDM allergen in a volume of 50 μl per mouse four times a week. In control groups, HDM allergen was replaced with 0.9% NaCl. Before HDM allergen application, mice were anesthetized with isoflurane vapor (3 to 3.5%). During the sensitization phase, the body weights of the mice were recorded once a week and additionally on days 56, 57, and 58 p.i. The final challenge with HDM allergen was performed on day 57 p.i., i.e., 24 h before the end of the animal experiment. On day 58 p.i., mice were subjected to AHR tests and sacrificed to obtain material for further analyses (Fig. 1A).

Fig 1.

Fig 1

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Experimental design, weight loss, and clinical scores in the infection and sensitization phases and clinical parameters used to assess acute pneumonia 5 days p.i. (A) Experimental design. Mice were infected i.n. with 1.3 × 107 IFU of C. pneumoniae; mock-treated controls received Chlamydia-free cell debris. Body weights (B) and clinical scores (C) were monitored daily during the first 4 weeks. The weights and elevated scores of the C. pneumoniae-infected (Cpn) groups nearly overlapped, while the mock- and not-treated groups also showed a not-distinguishable course almost identical to the basal levels. From day 28 p.i., mice were sensitized with 25 μg of HDM extract i.n. as indicated. Negative controls received identical volumes of NaCl. The final HDM allergen challenge was performed on day 57 p.i. On the following day (i.e., day 58), mice were subjected to AHR tests and then sacrificed to obtain samples for analyses. Two additional control groups (C. pneumoniae and mock infections) were sacrificed on day 5 to characterize acute lung infection. (D) Bacterial loads in the lungs of C. pneumoniae- and mock-infected mice 5 days p.i. IFU counts of C. pneumoniae were determined by titration of lung homogenate onto HeLa-T cell monolayers and subsequent (48 h) immunofluorescence analysis. Chlamydiae could not be detected in any of the mock-treated groups at either 5 or 58 days p.i. Additionally, infectious bacteria could no longer be found in the C. pneumoniae-treated groups at 58 days p.i. (E) Lung histological inflammatory scores (5 days p.i.) based on the following parameters: inflammatory cells, hemorrhages, edema, affected area, peribronchiolar infiltrates, and overall disease pathology score. (F) Cytokine profile in lung homogenate (5 days p.i.) determined by bead array analyses. Means ± the standard errors of the means are depicted. Asterisks indicate significant differences between infected and mock-infected mice (*, P < 0.05; ***, P < 0.001). MCP-1, monocyte chemoattractant protein 1.

Measurement of AHR.

Twenty-four hours after the last HDM allergen challenge, pulmonary function was measured invasively in anesthetized, spontaneously breathing mice (performed at the Fraunhofer Institute for Toxicology and Experimental Medicine [ITEM], Hannover, Germany) to assess AHR as described previously (38, 39). Briefly, pulmonary resistance (RL) was calculated from the measured differences in transpulmonary pressure and tidal respiratory flow by body plethysmography (type 871; HSE-Harvard Apparatus, March-Hugstetten, Germany) under isoflurane anesthesia in orotracheally intubated animals (by HEM 4.2 software; Notocord Systems, Croissy, France). After recording baseline values for 2 min, AHR to increasing doses of aerosolized methacholine chloride (MCh; Sigma, Deisenhofen, Germany) was determined by using a Bronchy type III generator (Fraunhofer ITEM) in combination with a feedback dose control system (38). Mice were exposed to MCh aerosol (from a 5% solution) in dose steps of 0.032, 0.063, 0.125, 0.3, 0.5, 1, 2, and 4 μg. Aerosol concentrations were determined with a gravimetrically calibrated photometer, and lung function was recorded continuously. From dose-effect plots (delta% RL versus MCh dose), the effective inhalation doses in micrograms of MCh required to produce 100, 150, and 200% increases in RL were evaluated for each animal (ED100, ED150, and ED200 RL). To obtain material for further analyses, mice were euthanized directly after lung function measurement by an overdose of Na-pentobarbital.

Preparation of lung homogenate and determination of the lung bacterial load.

Homogenate from right lungs was obtained as described previously (36). For the measurement of the bacterial burden by titration, thawed lung homogenate was serially diluted and centrifuged onto HeLa-T cell monolayers growing on cover slides. After 48 h, the slides were washed, fixed in ice-cold methanol, and stored at −20°C before staining with Chlamydia-specific antibody (Pathfinder Chlamydia culture confirmation system; Bio-Rad, Munich, Germany). Numbers of IFU per milliliter were determined by immunofluorescence microscopy (Axioskop; Zeiss, Jena, Germany).

Lung histopathology.

Lung histopathology was determined as described previously (36). Sections were stained with H&E (hematoxylin-and-eosin stain, Prisma-E2S; Sakura Tissue-Tek) or PAS (periodic acid-Schiff stain, Prisma-E2S; Sakura Tissue-Tek) and then analyzed by light microscopy (Axioskop 40; Zeiss, Jena, Germany). All tissue samples were graded by a blinded expert. For the determination of pathology typical of pneumonia, the degree of inflammation was scored in the H&E-stained sections according to the following parameters: inflammatory cells, hemorrhaging, edema, affected area, peribronchiolar infiltrates, and overall disease pathology score. Mouse lung pathology was determined by adding all of the points, resulting in a score of 0 (not affected) to 26 (highly affected) points.

To quantify mucus production in the airways, digitalized PAS-stained sections were used. The ratio of mucus-positive epithelium to total epithelium was calculated as the percentage of the surface covered in mucus (Adobe CS Photoshop Extended).

Determination of cytokine levels in lung homogenate and bronchoalveolar lavage fluid (BALF).

For the quantification of cytokines in lung tissue homogenate, the Mouse Inflammation Cytometric Bead Array (BD Biosciences, San Diego, CA) was used according to the manufacturer's instructions. During the preparation of lung homogenate, 1 μl protease inhibitor (Complete; Roche, Mannheim, Germany) was added to 100 μl of homogenate before freezing. Analysis was performed on a FACSCalibur (BD Biosciences, Franklin Lakes, NJ). For the quantification of mouse cytokines in BALF, the Mouse Th1/Th2 9-Plex Assay (for IFN-γ, TNF-α, KC/GRO, IL-1, IL-2, IL-4, IL-5, IL-10, and IL-12; Meso Scale Diagnostics, Gaithersburg, MD) was additionally used according to the manufacturer's instructions. Analysis was performed on a SECTOR Imager (Meso Scale Diagnostics, Gaithersburg, MD).

Enzyme-linked immunosorbent assays (ELISAs) for IgG, IgE, and C3a/C3a-desArg.

Whole blood was collected by heart puncture and incubated for 2 h at room temperature (RT). After centrifugation (10 min, 500 × g, RT), serum was collected and frozen at −80°C.

Determination of serum anti-Chlamydia IgG levels was performed as described previously (36). Ninety-six-well plates were coated with crude C. pneumoniae homogenate (1 μg/ml) as the antigen. Samples were diluted 1:10 to 1:5,000, depending on the experimental group and ELISA type. Horseradish peroxidase-linked antibodies were used for the detection of total IgG (115-036-062; Dianova, Hamburg, Germany), IgG1 (559626; BD PharMingen, Franklin Lakes, NJ), or IgG2a (553391; BD PharMingen) levels. Serial dilutions of pooled serum from C. pneumoniae-infected mice were used to obtain standard curves. Additionally, total IgG levels in preimmune serum samples collected before infection were determined exemplarily for each group.

For measurement of HDM allergen-specific IgE antibodies, plates were coated with 100 μl of 2 μg/ml anti-mouse IgE (R35-72; BD Biosciences, San Jose, CA) and incubated at RT for 4 h. Assay diluent buffer (BD Biosciences) was used to block plates. Serum samples were incubated in 1:2 titration steps overnight at 4°C, followed by digoxigenin-HDM allergen (made in our own laboratory) and digoxigenin-conjugated peroxidase (Hoffmann-La Roche, Basel, Switzerland) at RT, followed by tetramethylbenzidine substrate (Dako, Hamburg, Germany). The standard represents arbitrary (laboratory) units and was generated in house with serum samples obtained from BALB/c mice after repetitive (four times) intraperitoneal (i.p.) immunization with HDM allergen.

C3a/C3a-desArg, as markers of complement activation, were detected in undiluted BALF by sandwich ELISA with an anti-C3a neo epitope-specific capture antibody (558250; BD, Heidelberg, Germany) and a biotinylated anti-C3a (558251; BD, Heidelberg, Germany) detection antibody as described previously (36). For generation of standard curves, zymosan-activated EGTA plasma from C57BL/6J mice was used.

Cell profile in BALF.

After cannulation, airways were flushed twice with 0.8 ml ice-cold 0.9% NaCl. The absolute number of cells per milliliter of BALF was determined. BALF was centrifuged (10 min, 307 × g, 4°C), and 5 × 104 cells in 100 μl NaCl solution were subjected to centrifugation (700 rpm, RT, 5 min, Cellspin I; Tharmac, Waldsolms, Germany). Slides were stained with a Diff-Quick staining kit (Medion Diagnostics, Düdingen, Switzerland) according to the manufacturer's protocol. Three hundred cells per slide were counted in random fields by light microscopy to calculate the cellular distribution in BALF.

Group size and statistics.

The five 58-day groups (C. pneumoniae/HDM allergen treated, C. pneumoniae/NaCl treated, mock/HDM allergen treated, mock/NaCl treated, and not treated) consisted in most experiments of 16 to 23 animals per group. There were two exceptions. For the analysis of cellular distribution in BALF, 9 to 13 mice per group were used. Cytokines were determined in 10 animals per group. The two 5-day groups consisted of 11 infected (C. pneumoniae) and 6 mock-infected (mock) animals, respectively.

The results of the cytokine measurements (by Cytometric Bead Array and 9-Plex Assay) and of the C3a/C3a-desArg ELISA showed a Gaussian distribution. They were statistically analyzed by two- or one-way analysis of variance, respectively, with a Bonferroni post hoc test. For all other data that characterize experimental allergic asthma, in a first step, parameters were identified that are affected by HDM allergen sensitization. Only for those was the effect of C. pneumoniae on HDM allergen-induced AAD assessed statistically (Mann-Whitney test). The Student t test was used to analyze relative cell counts in BALF. For graphical and statistical analyses, GraphPad Prism V5 (GraphPad Software Inc., La Jolla, CA) was used.

RESULTS

To assess whether a preceding C. pneumoniae lung infection in BALB/c mice influences the severity and outcome of sensitization and challenge with the physiologic HDM allergen, mice were initially infected i.n. with chlamydial elementary bodies or mock infected as controls. Subsequently, at 28 days p.i., mice were sensitized and challenged with HDM allergen repetitively or with NaCl as a control for an additional ∼4 weeks (Fig. 1A).

C. pneumoniae induces severe pneumonia in mice, followed by recovery until sensitization with HDM allergen.

All three C. pneumoniae-infected groups showed drastic weight loss within 1 week p.i. (Fig. 1B); none of the mice died. In parallel, the infected mice exhibited an elevated clinical score reaching a maximum between days 3 and 7, indicating severe pneumonia (Fig. 1C). From day 8 on, C. pneumoniae-infected mice started to recover. On day 28 p.i., when the first sensitization with HDM allergen took place, C. pneumoniae-infected mice no longer showed clinical symptoms. During the sensitization phase, the body weights of the mice in all of the groups increased slightly but constantly and no clinical symptoms became apparent. In accordance with complete clinical recovery, no infectious bacteria could be detected at 58 days p.i. in any of the experimental groups.

Detailed characterization of acute pneumonia induced by C. pneumoniae was performed. For that purpose, groups of C. pneumoniae- or mock-infected mice were sacrificed on day 5 when clinical symptoms, i.e., loss of body weight and clinical score, were at their peaks. At 5 days p.i., infectious elementary bodies of C. pneumoniae could be detected only in the lungs of C. pneumoniae-infected mice at high levels of up to 107 IFU/ml and all of the animals in the noninfected control group were negative (Fig. 1D).

To confirm the infection status of the animals undergoing both phases of the experiment, Chlamydia-specific IgG levels were determined. Serum samples collected before infection from the mice in all of the groups were negative for Chlamydia-specific total IgG, as demonstrated for the not-treated group (Fig. 2A), confirming the Chlamydia-free status of the animal facility. As expected, on day 58 p.i., the levels of total Chlamydia-specific IgG, as well as of IgG1 and IgG2a, were elevated in serum samples from almost all of the C. pneumoniae-infected mice, in contrast to those in the serum samples of mock- or not-infected animals (Fig. 2). Intriguingly, the level of total C. pneumoniae-specific IgG in the C. pneumoniae/HDM allergen-treated group turned out to be significantly higher than the level in mice that had received only NaCl as a control after clinical recovery from pneumonia and during sensitization (C. pneumoniae/NaCl-treated group) (Fig. 2A). The levels of C. pneumoniae-specific IgG1 and IgG2a were also higher in mice that were sensitized to HDM allergen at 4 weeks p.i. However, these differences did not reach significance (Fig. 2B and C).

Fig 2.

Fig 2

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C. pneumoniae-specific IgG antibody levels at 58 days p.i. Total IgG (A), IgG1 (B), and IgG2a (C) levels in serum were determined. No Chlamydia-specific IgG could be detected in preimmune serum samples obtained from mice before infection (data not shown). Means ± the standard errors of the means are depicted. Asterisks indicate significant differences (**, P < 0.01).

Lung histology at day 5 p.i. revealed a high histological inflammatory score of ∼15 points only in the C. pneumoniae-infected group (Fig. 1E). Of note, there was still a slightly raised score in the C. pneumoniae/NaCl group 8 weeks after infection, which is 4 weeks after the disappearance of clinical symptoms (Fig. 3A). Nevertheless, on day 58 p.i., we found similarly elevated inflammatory histological scores in the lungs in the C. pneumoniae/HDM allergen- and mock/HDM allergen-treated groups (Fig. 3A).

Fig 3.

Fig 3

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Histological inflammatory scores, levels of IL-12 and KC/GRO, and complement activation in the lungs at 58 days p.i. (A) Lung histological inflammatory scores based on the following parameters: inflammatory cells, hemorrhaging, edema, affected area, peribronchiolar infiltrates, and overall disease pathology scores. For comparison: the score of mice infected with C. pneumoniae (Cpn) for 5 days reached ∼15 points. (B) Levels of IL-12 and KC/GRO in BALF. (C) C3a/C3a-desArg levels in BALF as a measure of complement activation. Means ± the standard errors of the means are depicted. Asterisks indicate significant differences (***, P < 0.001; n.s., not significant).

Cytokine profiles of lung homogenates proved severe lung inflammation, with elevated levels of IFN-γ, TNF-α, monocyte chemoattractant protein 1, IL-6, IL-10, and IL-12 in acute C. pneumoniae infection (day 5 p.i.) compared to those of the mock-treated group (Fig. 1F).

We also determined cytokine levels in BALF 24 h after the last HDM allergen challenge. In contrast to the results obtained on day 5 of acute C. pneumoniae lung infection, the concentrations of most of the cytokines (IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-5, and IL-10) determined at the end of the sensitization phase were close to or below the limit of detection. Exceptions were IL-12 and KC/GRO. Values in the mock/HDM allergen- and C. pneumoniae/HDM allergen-treated groups were significantly higher than those in the corresponding controls. Again, no difference between these two cytokines was found in the two HDM allergen-treated groups (Fig. 3B). Additionally, a similar increase in anaphylatoxin C3a, indicating complement activation, could be measured in both HDM allergen-treated groups (Fig. 3C).

Severity of experimental asthma is not increased after preceding chlamydial infection.

To address AHR as a central feature of asthma, a hyperresponsiveness assay (bronchial challenge test) was performed. Bronchoconstriction due to increasing doses of nebulized MCh was determined in which mice with AHR respond to lower doses of MCh. The effective doses (EDs) of MCh needed to produce 100, 150, and 200% increases in lung resistance above the baseline were calculated. The ED150s for the five long-term groups are depicted in Fig. 4A. The MCh EDs needed to elicit a lung resistance increase of 150% in the mock/NaCl-treated, not-treated, and C. pneumoniae/NaCl-treated groups were similar (in the range of 0.35 to 0.44 μg MCh). Both HDM allergen-treated groups were more sensitive to MCh and showed lower ED150s than the corresponding NaCl controls. Most important, comparison of the C. pneumoniae/HDM allergen-treated (0.18 μg MCh) group with the mock/HDM allergen-treated (0.1 μg MCh) group demonstrated that C. pneumoniae infection does not worsen the lung function of the C. pneumoniae/HDM allergen-treated group. On the contrary, this group turned out to be even slightly but significantly more resistant to MCh than the mock/HDM allergen-treated group. Similar results were obtained for the ED100s and ED200s (data not shown).

Fig 4.

Fig 4

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Lung function, HDM allergen-specific IgE level, and mucus secretion at 58 days p.i. (A) Effective inhalational dose in μg MCh required to produce a 150% increase in lung resistance (ED150 RL) above the baseline. Measurements of anesthetized, orotracheally intubated, and spontaneously breathing mice were performed. (B) HDM allergen-specific IgE levels measured in serum by ELISA. (C) Mucus production is the ratio of mucus-positive epithelium to total epithelium of 4-μm-thick, PAS-stained, formalin-fixed lung sections. Means ± the standard errors of the means are depicted. Asterisks indicate significant differences (*, P < 0.05; n.s., not significant).

Elevated allergen-specific IgE levels and increased mucus production in the airways are other typical features of allergic asthma. An ELISA was used to determine the HDM allergen-specific IgE titers in the serum of the mice (Fig. 4B). In order to measure mucus secretion, the percentage of mucus-positive bronchial epithelium was determined in PAS-stained lung sections and the ratio of mucus-positive epithelium to total epithelium was calculated (Fig. 4C). As expected, the HDM allergen-treated groups had significantly more HDM allergen-specific IgE in their serum and mucus in their airways than did the groups not treated with the allergen. Consistent with the results of lung function, C. pneumoniae infection increased neither the IgE titers nor the mucus secretion of the C. pneumoniae/HDM allergen-treated group compared to that of the mock/HDM allergen-treated group. Rather, a slight but not significant reduction in both parameters was observed in the C. pneumoniae/HDM allergen-treated mice (P = 0.36 for IgE levels, P = 0.14 for mucus production).

The cellular distribution in BALF was determined in cytospin preparations and by H&E staining. Treatment with HDM allergen led to a significant increase in the total cell numbers in BALF (Fig. 5A). A small but not significant (P = 0.099) increase in absolute cell numbers was observed in fluids from C. pneumoniae/HDM allergen-treated mice compared to those in fluids from mock/HDM allergen-treated animals. More importantly, the relative cellular distribution was significantly different between these two groups (Fig. 5B). Infection with C. pneumoniae did not augment the proportion of eosinophils, which are characteristic of bronchial asthma. On the contrary, significantly fewer eosinophils (∼20% versus ∼45%) were present in the BALF of C. pneumoniae/HDM allergen-treated mice than in the BALF of mock/HDM allergen-treated mice. Enumeration of the cells in the BALF of C. pneumoniae/HDM allergen-treated mice revealed much higher proportions of alveolar macrophages (∼55% versus ∼38%) and lymphocytes (∼9% versus ∼2%) than in that of the mock/HDM allergen-treated group. No significant differences in neutrophil numbers were found.

Fig 5.

Fig 5

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Cellular distribution in BALF on day 58 p.i. CytoSpot slides with 5 × 104 cells were prepared from BALF. Slides were stained with the Diff-Quick staining kit. Three hundred cells were counted per slide from random fields. (A) Total cell numbers in BALF. (B) Relative cell numbers in BALF; only the C. pneumoniae/HDM allergen- and mock/HDM allergen-treated group results are shown. Means ± the standard errors of the means are depicted. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant).

DISCUSSION

In this model, acute severe pneumonia was induced within 1 week after the i.n. application of C. pneumoniae, as indicated by body weight, clinical score, histopathology, and increased proinflammatory and Th1 cytokine levels. On the basis of clinical parameters and supported by control experiments where viable chlamydiae could no longer be recovered after 4 weeks p.i. and inflammatory marker levels in lung homogenate were no longer elevated, mice had recovered almost completely when sensitization with HDM antigen began. The only detectable residual sign of pneumonia at later time points was a slightly but significantly elevated histopathological inflammatory score after 4 weeks and even at 58 days p.i. compared to that of mock-infected animals. This indicates mild but long-lasting consequences of chlamydial lung infection and some degree of ongoing inflammation at the time point when sensitization with HDM allergen started.

In our model, we applied the allergen several times per week in a relatively low dose similar to that used by Al-Garawi et al. in the context of infection with other pathogens (40). From our point of view, this resembles the physiological situation in “real life” more closely.

The outcome of our experiments combining infection and sensitization clearly demonstrates that a preceding lung infection with C. pneumoniae did not increase the severity of HDM allergen-induced experimental asthma in mice. On the contrary, characteristics of AAD, like AHR and eosinophilia, were significantly diminished upon bacterial infection. Determination of AHR may be very sensitive because the parameter ED150 RL is calculated from a rather robust set of measurements describing a dose-response curve. Mucus secretion into the airways and HDM allergen-specific IgE levels were also reduced in mice that had suffered from pneumonia before sensitization; however, the observed differences did not reach significance.

Several factors may influence the development of asthma following an infection, i.e., the type of infection (pathogen), the time point of infection (neonatal, infant, adult), and the type of antigen.

So far, many animal models dissecting the interplay of Chlamydia and AAD have made use of the strict mouse pathogen C. muridarum: As demonstrated by Horvat et al. for ovalbumin as an allergen, infection of 6-week-old mice did not affect the typical features of subsequent experimental asthma (29, 30). Interestingly, infection of neonatal or infant (3-week-old) mice attenuated eosinophilia and ovalbumin-specific Th2 cytokine release on the one hand but on the other led to a mixed T cell response with aggravation of other hallmarks of asthma. The findings of Schroeder et al., who used HSA as an antigen, indicate that the time point and severity of C. pneumoniae infection also influence the kind of AAD that occurs. The parallel application of UV-inactivated or viable C. pneumoniae (or UV-inactivated Bordetella bronchiseptica) combined with regulatory T cells (Treg) and dendritic cell depletion experiments and the investigation of TLR4−/− and TLR2−/− mice indicate that Toll-like receptor 4 signaling is required for antigen sensitization. These results also indicate that chlamydial infection downregulates Treg cells and dendritic cells in a dose-dependent fashion, thereby modifying the allergic response (31, 32). Modification of the amount of Treg cells might also play a role in our model: Suppression of pathogenic Th2 responses by Treg cells may be impaired in asthmatic mice (even if the cytokine responses were below the limit of detection) and a high-dose infection with C. pneumoniae might counteract the proposed mechanism.

Allergens like ragweed and HDM allergen may be more representative of the physiological situation than ovalbumin or HSA (i.p./i.n.) is. By administering ragweed to 7- to 10-week-old mice, Bilenki et al. demonstrated that a preceding infection with C. muridarum is able to suppress allergic airway inflammation (41). A pattern with reduced eosinophilia similar to that observed in our experiments emerged. In addition, allergen-driven Th2 cytokine levels were diminished in that model. However, C. muridarum significantly increased the total IgE level whereas a slight but nonsignificant reduction of the HDM allergen-specific IgE titer in the C. pneumoniae/HDM allergen-treated group compared to that in the mock/HDM allergen-treated group became apparent in our study with C. pneumoniae.

We chose the human pathogen C. pneumoniae for our model (35, 36) in order to more closely mimic the situation in humans. Considering AHR, eosinophilia, mucus secretion, and HDM allergen-specific IgE, C. pneumoniae led to a similar or even slightly milder course of AAD induced by HDM allergen in 6-week-old animals (at the start of the experiment). These results are in accordance with the findings of Bilenki et al. obtained with ragweed and C. muridarum (41) and are compatible with the results obtained by Schroeder et al. by using higher doses of C. pneumoniae and applying the antigen HSA at later time points p.i (31). Using ovalbumin and C. muridarum in adult mice, Horvat et al. found results similar to ours, but those findings differed for newborn and infant mice, at least with regard to mucus secretion and AHR. This demonstrates the complexity of the relationship between respiratory pathogens and allergic lung disease. It also suggests that the timing of infection might influence the outcome of the allergic response.

Our findings are in good agreement with studies on other pathogens. Infection with Streptococcus pneumoniae or treatment with killed streptococci suppressed pulmonary eosinophil accumulation, AHR, and antigen-specific Th2 cytokines (42). Lung infection of C57BL/6 mice with Mycobacterium bovis induced a decrease in lung eosinophilia, an increase in IFN-γ, and a reduction of IL-5 during subsequent Cryptococcus infection (43). M. pneumoniae infection prior to sensitization with ovalbumin suppresses airway inflammation and AHR (44). Even Helicobacter pylori, a pathogen of the intestinal tract, protects mice efficiently from AHR, tissue inflammation, and goblet cell metaplasia and prevents ovalbumin- or HDM allergen-induced pulmonary and bronchoalveolar infiltration with eosinophils, Th2 cells, and Th17 cells (45).

As the hygiene hypothesis is based on the interaction between the infectious Th1 response and the allergic Th2 response, it is highly informative to investigate this interplay in suitable animal models. Unfortunately, it was impossible to compare the Th1 and Th2 cytokine patterns in detail after the last challenge with HDM allergen because of sensitivity issues. We could only detect large amounts of the Th1 cytokine IL-12 and the neutrophil chemoattractant KC/GRO in BALF but without any significant difference between the C. pneumoniae/HDM allergen- and mock/HDM allergen-treated groups.

To our surprise, the total C. pneumoniae-specific IgG level observed at day 58 p.i. was significantly higher if infected mice were additionally sensitized to HDM allergen from day 28 p.i. on. Thus, the immune or inflammatory responses in AAD can augment an ongoing humoral response to C. pneumoniae at a time point when the lung infection is almost completely resolved and only a few signs of inflammation are still detectable. This effect might be caused by a mediator that is participating in both chlamydial infection/immunity and AAD. One example of such a mediator is C3a. On the one hand, the complement system is activated during acute chlamydial lung infection (36). On the other hand, complement is activated and the C3a level is also elevated in AAD (6), as confirmed by our data obtained on day 58. As recently reviewed by Heeger and Kemper (46), C3a can augment antigen presentation and T cell responses to various pathogens. Additionally, C3b (which must be generated in parallel during complement activation) binds to antigens, thereby enhancing the humoral response. One can therefore speculate that the cleavage products of C3 generated during the AAD phase of our model amplify, as a bystander effect, an ongoing humoral response to Chlamydia.

C. pneumoniae-specific IgE was also checked by basically following a protocol successfully used by Bilenki et al. for C. muridarum (47) after adaptation for C. pneumoniae. However, no C. pneumoniae-specific IgE was detectable in the C. pneumoniae/HDM allergen or the C. pneumoniae/NaCl group (data not shown).

In conclusion, our results indicate that a severe preceding clinically resolved lung infection with C. pneumoniae does not increase the severity of AAD. On the contrary, C. pneumoniae even led to a slightly milder course of experimental asthma induced by HDM allergen.

However, it should be noted that our data do not exclude the possibility that the situation is different in neonatal or infant mice. Moreover, temporal patterns of exposure to HDM allergen and to C. pneumoniae, as well as the quantity and frequency of exposure and the severity of the preceding lung infection, may also have a pivotal influence on the outcome of AAD. Furthermore, two additional possibilities might account for the epidemiological relationship found between C. pneumoniae and asthma, i.e., (i) that pneumonia increases the incidence of an allergic response and (ii) that preexisting AAD is influenced by subsequent C. pneumoniae infection. To address these questions, future experiments in a different experimental setting with animal models and longitudinal epidemiological studies with accurate parameters for exposure and response to the agents in humans have to be performed.

ACKNOWLEDGMENTS

This project was financially supported by DFG-funded CRC587 (Immune Reactions of the Lung in Allergy and Infection): A.B., B4; A.K. and B.F., A16; A.-M.D., N01; H.G.H., Z2. It was also partially supported by DFG-funded European Research Training Group 1273 (Strategies of Human Pathogens to Establish Acute and Chronic Infections) (P.D.). There are no competing interests.

We thank G. Bartling, S. Pourebrahim, and E. Wiebe of Hannover Medical School and S. Dunker and D. Schellbach of ITEM for their excellent help as technical assistants and S. Suerbaum for his support.

P.D. participated in the handling of mice in the animal model (infection, scoring, weighing, collection of tissue and samples, and their preparation for analysis) and carried out all analyses, with the exception of histology, anti-HDM allergen IgE measurement, BALF cell counting, and lung function. Moreover, P.D. was involved in the overall design of the study with a focus on mouse infection. He performed data acquisition, calculations, and statistical analysis (with the exception of lung function) and wrote the manuscript together with A.K.. S.L. participated in the collection of samples and performed BALF analysis. R.L. participated (as a technician) in the handling of mice in the animal model (infection, scoring, weighing, collection of tissue and samples, and their preparation for analysis). Moreover, he was involved in the overall design of the study with a focus on mouse infection. S.G. prepared and stained lung sections and performed microscopy and histological scoring, including determination of mucus production. Additionally, she helped to draft the corresponding part of the manuscript. H.G.H. participated in the design and development of the HDM allergen sensitization procedure and conducted lung function measurement and analysis, including statistics. Additionally, he helped to draft the corresponding part of the manuscript. A.-M.D. participated in the overall design and development of the HDM allergen sensitization procedure, performed HDM allergen-specific IgE analysis, and helped to draft the manuscript. B.F. participated in the determination of C. pneumoniae-specific antibodies and the handling of mice in the animal model. M.M. participated in the design of the study and the handling of mice in the animal facility. Additionally, she helped in the cytokine analysis by Mouse Th1/Th2 9-Plex Assay. A.B. participated in the overall design of the study with a focus on allergic disease and helped to draft the manuscript. A.K. conceived of the study and was involved in its overall design. He helped in data and statistical analyses (with the exception of lung function), wrote the manuscript together with P.D., and coordinated the study. All of us read and approved the final manuscript.

Footnotes

Published ahead of print 1 July 2013

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