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Published ahead of print on December 30, 2004, doi:10.1165/rcmb.2004-0197OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 201-210, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0197OC

Mycoplasma pneumoniae Induces Host-Dependent Pulmonary Inflammation and Airway Obstruction in Mice

Monica Fonseca-Aten, Ana M. Ríos, Asunción Mejías, Susana Chávez-Bueno, Kathy Katz, Ana M. Gómez, George H. McCracken, Jr. and R. Doug Hardy

Departments of Pediatrics, Internal Medicine, and Pathology, University of Texas Southwestern Medical Center, Dallas, Texas

Correspondence and requests for reprints should be addressed to Monica Fonseca-Aten, M.D, Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9063. E-mail address: Monica.Fonseca-Aten{at}utsouthwestern.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Respiratory tract infections result in wheezing in a subset of patients. Mycoplasma pneumoniae is a common etiologic agent of acute respiratory infection in children and adults that has been associated with wheezing in 20–40% of individuals. The current study was undertaken to elucidate the host-dependent pulmonary and immunologic response to M. pneumoniae respiratory infection by studying mice with different immunogenetic backgrounds (BALB/c mice versus C57BL/6 mice). After M. pneumoniae infection, only BALB/c mice developed significant airway obstruction (AO) compared with controls. M. pneumoniae–infected BALB/c mice manifested significantly elevated airway hyperresponsiveness (AHR) compared with C57BL/6 mice 4 and 7 d after inoculation as well as BALB/c control mice. Compared with C57BL/6 mice, BALB/c mice developed worse pulmonary inflammation, including greater peribronchial infiltrates. Infected BALB/c mice had significantly higher concentrations of tumor necrosis factor-{alpha}, interferon-{gamma}, interleukin (IL)-1ß, IL-6, IL-12, KC (functional IL-8), and macrophage inflammatory protein 1{alpha} in the bronchoalveolar lavage fluid compared with infected C57BL/6 mice. No differences in IL-2, IL-4, IL-5, IL-10, and granulocyte/macrophage colony-stimulating factor concentrations were found. The mice in this study exhibited host-dependent infection-related AO and AHR associated with chemokine and T-helper type (Th)1 pulmonary host response and not Th2 response after M. pneumoniae infection.

Key Words: Mycoplasma pneumoniae • airway obstruction • asthma • airway hyperresponsiveness • cytokines • chemokines


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The appearance of wheezing in association with acute respiratory tract infections with viruses, Mycoplasma pneumoniae, and Chlamydophila pneumoniae involves a subset of infected patients; thus, it has been postulated that these respiratory pathogens trigger the wheezing process in subjects who are predisposed as a result of their genetic background (1–4). The immunopathogenic mechanisms that result in the variable induction of the wheezing phenotype by respiratory tract infections in a host are not well defined.

M. pneumoniae is a common cause of respiratory tract infection in children and adults accounting for as many as 20–40% of all cases of community-acquired pneumonia (2, 5–7). Recently, it has become more strongly associated with wheezing syndromes and asthma. There is growing evidence linking M. pneumoniae infection and the inception, exacerbation, and chronicity of asthma (8–12). The presence of M. pneumoniae has been detected in up to 20–29% of patients with acute asthma exacerbations (2, 10, 13, 14). In addition, wheezing has been consistently documented in 20–40% of children with acute M. pneumoniae respiratory infection (2, 15).

A murine model of M. pneumoniae respiratory infection that closely resembles human M. pneumoniae respiratory disease has been previously established (16–18). In this model, M. pneumoniae causes both acute and chronic respiratory infection in BALB/c mice with associated pulmonary inflammation and pulmonary function abnormalities (airway obstruction [AO] and airway hyperresponsiveness [AHR]). The inflammatory response is manifested by abnormal pulmonary histopathology and elevated concentrations of cytokines and chemokines in bronchoalveolar lavage (BAL) specimens (16, 19, 20).

Host immunogenetic heterogeneity has been shown to determine the nature and susceptibility to infectious diseases in humans (21). On the basis of the clinical reports suggesting that M. pneumoniae causes host selective disease, the current study was undertaken to investigate the variable pathophysiologic and immunologic host response to M. pneumoniae respiratory infection by using two strains of inbred mice with different immunogenetic background (BALB/c mice versus C57BL/6 mice). These two mouse strains have been reported to develop different immune responses following infection with various bacteria (22–24), viruses (25, 26), and parasites (27, 28). Furthermore, they have differences in their histocompatibility loci (H-2 region; BALB/c mice carry the H-2d locus and C57BL/6 mice carry the H-2b locus) and in other H-2–associated genes, mimicking differences among humans (24, 29, 30).

We found host dependent pulmonary inflammation, AO, and AHR in response to M. pneumoniae respiratory infection. In addition, each mouse strain exhibited a distinct pulmonary chemokine and cytokine profile.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Organism and Growth Conditions
M. pneumoniae (ATCC 29342; ATCC, Manassas, VA) was reconstituted in SP4 broth and subcultured after 24–48 h in a flask containing 20 ml of SP4 media at 37°C. When the broth turned an orange hue (~ 72 h), the supernatant was decanted, and 2 ml of fresh SP4 broth was added to the flask. A cell scraper was used to harvest the adherent mycoplasmas from the bottom of the flask. This achieved a M. pneumoniae concentration in the range of 108–109 colony-forming units (cfu)/ml. Aliquots were stored at –80°C. All SP4 media contained nystatin (50 U/ml) and ampicillin (1.0 mg/ml) to inhibit growth of potential contaminants.

Animals and Inoculation
Mice were obtained from a commercial vendor (Charles River Laboratories, Wilmington, MA), who confirmed their mycoplasma- and murine virus–free status. Mice were housed in the animal care facility of our institution in filter-top cages in a temperature-controlled room (22°C) and allowed to acclimate to their new environment for 1 wk. The Animal Resource Center at the University of Texas Southwestern Medical Center performed quarterly health surveillance on sentinel mice housed in the mouse storage room. Sentinel mice were examined for antibodies against mouse hepatitis virus, Sendai virus, pneumonia virus of mice, reo-3 virus, mouse encephalitis virus (GD-7), mouse rotavirus (EDIM), minute virus of mice, and Mycoplasma pulmonis, and were also screened for pinworm and mites. Sentinel mice tested negative for these pathogens. Methoxyflurane, an inhaled anesthetic, was used for sedation during inoculation. Two-month-old female BALB/c mice and C57BL/6 mice were intranasally inoculated once (Day 0) with 0.8 x 107–1.5 x 107 cfu of M. pneumoniae in 50 µl of SP4 broth. Directly comparable groups were given inoculum from the same batch. Control BALB/c and C57BL/6 mice were inoculated with sterile SP4 broth. All mice were housed in the same animal room and received identical daily care. Animal guidelines were followed in accordance with the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center at Dallas.

Experimental Design and Sample Collection
Mice were evaluated at 1, 4, and 7 d after inoculation. Samples were obtained from eight to nine mice per group (BALB/c mice, infected and control; C57BL/6 mice, infected and control) at each time point from repeated experiments; whole-body, unrestrained, nonsedated plethysmography was performed in 12–16 mice per group at each time point. Mice were anesthetized with an intraperitoneal injection of 75 mg/kg ketamine and 5 mg/kg acepromazine before cardiac puncture. Blood was centrifuged at 3,500 x g for 10 min, and the plasma was stored at –80°C. Bronchoalveolar lavage (BAL) specimens were obtained by instilling 500 µl of SP4 broth through a 25-gauge needle into the lungs, via the trachea, followed by aspiration of this fluid into a syringe. Approximately 70–80% of the instilled volume was consistently retrieved. All BAL samples were kept on ice until processed. Whole lung specimens, including the trachea and both lungs, were collected and fixed with a 10% buffered formalin solution for histologic evaluation.

Culture
Twenty-five microliters of undiluted sample and serial 10-fold dilutions of BAL fluid in SP4 broth (50 µl of undiluted sample was used for the initial dilution) were immediately cultured on SP4 agar plates at 37°C, whereas the remaining undiluted BAL specimens were stored at –80°C. Quantification was performed by counting colonies on plated specimens and expressed as log10 cfu/ml.

Histopathology
Lung tissue was fixed in buffered formalin, and transverse sections were stained with hematoxylin and eosin. The histopathologic score (HPS) was determined by a single pathologist who was unaware of the mouse strain and infection status of the animals from which specimens were taken. The HPS was based on grading of peribronchiolar and bronchial infiltrate, bronchiolar and bronchial luminal exudate, perivascular infiltrate, and parenchymal pneumonia (neutrophilic alveolar infiltrate). This HPS system assigned values from 0–26: the higher the score, the greater the inflammatory changes in the lung (16–18, 31). The extent of variation in HPS when the same slide is scored by the same pathologist multiple times has been found to be 0 to 1.

Measurement of Cytokines and Chemokines in BAL and Sera
Concentrations of cytokines and chemokines in BAL specimens and concentrations of cytokines in sera were assessed using Multiplex Bead Immunoassays (BioSource International, Camarillo, CA) in conjunction with the Luminex LabMAP system, following the manufacturer's instructions. The cytokines and chemokines examined and their levels of sensitivity were as follows: tumor necrosis factor-{alpha} (TNF-{alpha}), 5 pg/ml; interferon (IFN)-{gamma}, 1 pg/ml; interleukin (IL)-1ß, 10 pg/ml; IL-2, 15 pg/ml; IL-4, 5 pg/ml; IL-5, 10 pg/ml; IL-6, 10 pg/ml; mouse KC (functional IL-8), < 15 pg/ml; IL-10, 15 pg/ml; IL-12 (p40/p70), 15 pg/ml; granulocyte/macrophage colony-stimulating factor (GM-CSF), 10 pg/ml; monocytes chemotactic protein (MCP)-1, < 5pg/ml; and macrophage inflammatory protein (MIP)-1{alpha}, < 15 pg/ml. For statistical analysis, samples with readings below the limit of the standard curve of the assay were assigned a value one half that of the lowest detectable value.

Whole Lung RNA Extraction and Analysis
Whole lung samples were collected from two to three mice per group 4 d after inoculation with M. pneumoniae or SP4 broth and stored in RNAlater RNA stabilization reagent (Qiagen, Valencia, CA). Samples were homogenized in RNazol reagent (Teltest, Friendswood, TX) and extracted with chloroform (IBI, New Haven, CT); total RNA was precipitated with isopropanol (Sigma-Aldrich, St. Louis, MO). The RNeasy mini-kit was used with on column DNase digestion RNase-free DNase (Qiagen) to remove traces of genomic DNA. RNA samples with absorbance at 260 nm:absorbance at 280 nm ratios of 1.8–2.1 were used for real-time reverse-transcriptase PCR. RNA concentration was measured spectrophotometrically and sample RNA integrity was confirmed by agarose gel electrophoresis. Detection of IL-4, IL-5, IL-12p40, TNF-{alpha}, IFN-{gamma}, and ß-actin mRNA levels was performed by real-time RT-PCR assay using an ABI prism 7700 Sequence detection system (PerkinElmer Biosystems, Boston, MA).

Reverse transcription of total RNA (2 µg) from whole lung cells was performed using the TaqMan Gold RT-PCR kit (ABI, Foster City, CA) in a 100-µl reaction following the manufacturer's instructions. For every reaction set, one RNA sample is performed without Multiscribe Reverse Transcriptase (RT-minus reaction) to provide a negative control in the subsequent PCR assays. Real-time PCR Primers and Fret Probes sets for IL-4, TNF-{alpha}, IFN-{gamma}, and ß-actin were obtained from BioSource International (Camarillo, CA) and used according to the manufacturer's instructions. Cytokines IL-12p40 and IL-5 TaqMan pre-developed assay reagents were obtained from ABI and used according to the instructions provided.

PCR amplification of whole lung cDNA samples (5 µl) was performed in a 50-µl reaction mix using the 2x ABI TaqMan Universal Master Mix. All samples were run in duplicate. The PCR amplification thermocyle profile cycling conditions were 95°C for 10 min for the Amplitaq-gold enzyme activation followed by 40 cycles with 15 s at 95°C for denaturing and 1 min at 60°C for annealing and extension. Both a PCR control (–cDNA) and the RT-minus reaction were run as negative controls for the PCR assays. Positive controls supplied with the Primer/Probes sets were also run in the assays. An endogenous control assay for ß-actin housekeeping gene was also run with all other cytokine assays for normalization. The threshold cycle (Ct), the fractional cycle number at which the amplified target reaches a significant threshold, was subsequently determined. Relative quantitation of IL-4, IL-5, IL-12p40, TNF-{alpha}, and IFN-{gamma} mRNA expression was evaluated using the comparative Ct method described elsewhere (32). The relative quantitation of the target value, normalized to an endogenous control ß-actin gene, and relative to a calibrator (controls), is expressed as 2-{Delta}{Delta}Ct (fold induction over the controls).

Plethysmography
Whole-body, unrestrained, nonsedated plethysmography (Buxco, Troy, NY) was used to monitor the respiratory dynamics of mice in a quantitative manner at baseline (AO), and after methacholine exposure (AHR). Before methacholine exposure, mice were allowed to acclimate to the chamber and then plethysmography readings were recorded to establish baseline values. Next, mice were exposed once to aerosolized methacholine (50 mg/ml); after exposure, plethysmography readings were recorded. Enhanced Pause (Penh) is a dimensionless value that represents a function of the ratio of peak expiratory flow to peak inspiratory flow and a function of the timing of expiration. Penh correlates with pulmonary airflow resistance or obstruction. Penh as measured by plethysmography has been previously validated in animal models of AHR (33–36).

Statistics
For all statistical analysis Sigma Stat 2003 software (SPSS Science, San Rafael, CA) was used. The t test was used to compare values of the different groups of animals at the same time point if the data were normally distributed. In the instances in which the data were not normally distributed, the Mann-Whitney rank sum test was used for comparisons. Bonferroni correction was used in the instances where multiple comparisons were made. The Spearman rank order test was used for correlations, as all the data taken together were not normally distributed. A comparison was considered statistically significant if the P value was < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Clinical Findings
The fur of all the BALB/c mice developed a ruffled appearance 1–2 d after inoculation with M. pneumoniae that persisted for ~ 2 d. No visual differences could be detected between the experimental and control C57BL/6 mice.

Lung Histopathology
Both BALB/c and C57BL/6 mice developed acute histologic inflammation after inoculation with M. pneumoniae compared with their respective controls. However, the BALB/c mice manifested more severe pulmonary inflammation compared with C57BL/6 mice as evidenced by significantly higher lung HPS at all time points evaluated (Figure 1). Figure 2 demonstrates the histopathologic appearance of a representative BALB/c mouse lung compared with that of a representative C57BL/6 mouse lung 4 d after inoculation with M. pneumoniae. The abnormal histopathologic findings in the BALB/c mouse consisted of peribronchial and perivascular dense circumferential mononuclear infiltrates and parenchymal pneumonia (neutrophilic alveolar infiltrate obliterating the airspaces). The C57BL/6 mouse lung demonstrated perivascular mononuclear infiltrates, but there were no peribronchial or alveolar infiltrates.



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  		Figure 1. Lung histopathologic score (HPS) of mice inoculated with M. pneumoniae (Mp) or sterile SP4 broth (controls). Values shown are the medians and the 25–75 percentiles (error bars). *P < 0.05 between the HPS of BALB/c mice infected with Mp and C57BL/6 mice infected with Mp. Bars represent results from two independent experiments, each including four to five mice per time point in each group.

 


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  		Figure 2. Comparative histopathologic appearance (magnification: x20) of (A) BALB/c mouse lung and (B) C57BL/6 mouse lung, 4 d after inoculation with M. pneumoniae showing more severe inflammation in the infected-BALB/c compared with the infected-C57BL/6 mouse.

 
AO
Although both mouse strains developed acute histologic inflammation in response to infection with M. pneumoniae, only BALB/c mice developed significant AO, as demonstrated by elevated baseline Penh compared with their respective controls (Figures 3A and 3B). Penh peaked at Day 1 in M. pneumoniae–infected BALB/c mice and was significantly elevated (P < 0.05) compared with the control groups at all time points.



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  		Figure 3. AO was assessed by whole-body plethysmography by measuring Penh in (A) BALB/c mice inoculated with Mp or sterile SP4 broth (controls) and (B) C57BL/6 mice inoculated with Mp or sterile SP4 broth (controls). Values shown are the medians and 25–75 percentiles (error bars). *P < 0.05 between mice infected with Mp and control mice. Values represent results from two independent experiments (n = 12–16 mice per time point per group).

 
AHR
Penh after standardized methacholine challenge was significantly elevated (P < 0.05) in the M. pneumoniae–infected BALB/c mice compared with that in controls at all time points evaluated (Figure 4A). Compared with controls, M. pneumoniae–infected C57BL/6 mice had a modest but significant increase in Penh after methacholine challenge on Days 1 and 4 after inoculation (Figure 4B). However, the infected BALB/c mice manifested significantly greater AHR compared with C57BL/6 mice by comparison of delta Penh values, which represent the difference between the Penh after methacholine challenge and the baseline Penh for each mouse (Figure 5).



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  		Figure 4. AHR was assessed by whole-body plethysmography by measuring Penh after methacholine challenge in: (A) BALB/c mice inoculated with Mp or sterile SP4 broth (controls) and (B) C57BL/6 mice inoculated with Mp or controls. Values shown are the medians and 25–75 percentiles (error bars). *P < 0.05 between mice infected with Mp and control mice. Values represent results from two independent experiments (n = 12–16 mice per time point per group).

 


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  		Figure 5. To assess AHR, mice were challenged with aerosolized methacholine and the changes in Penh recorded (Delta Penh). Values shown are the medians and 25–75 percentiles (error bars). *P < 0.05 between the Delta Penh of BALB/c mice infected with Mp and C57BL/6 mice infected with Mp. Values represent results from two independent experiments (n = 12–16 mice per time point per group).

 
M. pneumoniae Quantitative BAL Culture
BAL cultures were positive in 100% of the mice from both strains after inoculation with M. pneumoniae. BALB/c mice had higher quantitative M. pneumoniae BAL cultures than did C57BL/6 mice at all time points evaluated (Figure 6). The percent decline in quantitative BAL cultures between 1 and 7 d after inoculation was 10.94% for the BALB/c mice and 22.66% for the C57BL/6 mice, suggesting a faster clearance of the organism burden for mice of the C57BL/6 strain. All control mice had negative BAL cultures.



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  		Figure 6. Quantitative Mp cultures in BAL samples of infected BALB/c mice and C57BL/6 mice. Values shown are the means ± SD (error bars). Bars represent results from two independent experiments, each including four to five mice per time point in each group (*P < 0.05).

 
BAL Cytokines and Chemokines
The M. pneumoniae–infected BALB/c mice had significantly higher (P < 0.05) BAL concentrations of TNF-{alpha}, IFN-{gamma}, IL-1ß, IL-6, and IL-12 compared with their controls at one or more time points (Table 1). KC (functional mouse IL-8), MIP-1{alpha}, and JE/MCP-1 concentrations were significantly higher in infected BALB/c mice compared with controls (P < 0.05) (Figures 7A–7C). The infected C57BL/6 mice had significantly higher levels of TNF-{alpha}, IL-6, IL-12, KC, MIP-1{alpha}, and JE/MCP-1 compared with controls (Table 1, Figures 7A–7C).


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  		TABLE 1. Cytokine concentrations in bronchoalveolar lavage specimens from mice inoculated with mycoplasma pneumoniae or sterile SP4 broth (controls)

 




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  		Figure 7. Chemokine concentrations in BAL specimens from mice inoculated with Mp or sterile SP4 broth (controls) measured 1 d after inoculation. Values represent results from two independent experiments, each including four to five mice per time point in each group. Values shown are the medians and 25–75 percentiles (error bars). {dagger}P < 0.05 between the values for BALB/c mice inoculated with Mp and controls. {ddagger}P < 0.05 between the values for C57BL/6 mice inoculated with Mp and controls. *P < 0.05 between the values for BALB/c mice infected with Mp and C57BL/6 mice infected with Mp. Multiple comparisons were made with Bonferroni correction.

 
When comparing M. pneumoniae–infected mice of both strains, infected BALB/c mice had significantly higher BAL concentrations of TNF- {alpha}, IFN-{gamma}, IL-1ß, IL-6, IL-12, KC, and MIP-1{alpha} compared with infected C57BL/6 mice at one or more of the time points evaluated (Table 1, Figure 7).

The concentrations of IL-4 (Table 1), IL-5 (Table 1), IL-2, IL-10, and GM-CSF in BAL specimens were not statistically different when comparing infected BALB/c mice and C57BL/6 mice with their respective controls at any time point.

Real-Time RT-PCR Measurement of IL-4, IL-5, IL-12p40, TNF-{alpha}, and IFN-{gamma} in Whole Lung Samples
The mean relative expression of TNF-{alpha}, IFN-{gamma}, and IL-12p40 mRNA measured in lung tissue homogenates 4 d after inoculation was significantly higher in M. pneumoniae infected BALB/c mice relative to uninfected control BALB/c mice (P < 0.005) (Table 2). M. pneumoniae–infected C57BL/6 mice had higher lung tissue expression of IL-12p40 and TNF-{alpha} relative to uninfected strain-matched controls (5.12- and 2.70-fold induction over controls, respectively), but the difference was not statistically significant (Table 2). No difference in the relative expression of IFN-{gamma} was found in lung tissue samples of infected C57BL/6 mice relative to uninfected controls (Table 2). For both strains of mice no significant differences in the mean relative expression of IL-4 and IL-5 mRNA were found (Table 2).


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  		TABLE 2. Cytokine mRNA detected by real-time rt-pcr in lung homogenate specimens from mice inoculated with mycoplasma pneumoniae or sterile SP4 broth (controls) measured 4 d after inoculation

 
Systemic Cytokine Response
The concentrations of cytokines in serum were measured one day after inoculation. IL-6 and IL-12 concentrations were modestly but significantly elevated in infected BALB/c and C57BL/6 mice compared with their respective controls. BALB/c mice had a median serum IL-6 concentration of 137.9 pg/ml (range, 44.9–266.1 pg/ml) for the infected group versus 24.9 pg/ml (range, 10.0–46.0 pg/ml) for the control group (P < 0.001). C57BL/6 mice had a median IL-6 concentration of 50.4 pg/ml (range, 10.0–111.2 pg/ml) and 10.0 pg/ml (range, 10.0–46.0 pg/ml) for the infected and control groups, respectively (P = 0.033). Infected BALB/c mice had a median IL-12 concentration of 518.6 pg/ml (range, 311.6–744.2 pg/ml) compared with 433.7 pg/ml (range, 255.6–510.5 pg/ml) for controls (P = 0.047). For infected C57BL/6 mice, median concentration of IL-12 was 425.5 pg/ml (range, 329.7–523.7 pg/ml) mice versus 314.8 pg/ml (range, 161.0–359.4 pg/ml) for the control group (P = 0.002). However, no differences in the serum concentrations of TNF-{alpha}, IFN-{gamma}, IL-1ß, IL-2, IL-4, IL-5, IL-10, or GM-CSF were found when comparing M. pneumoniae–inoculated BALB/c and C57BL/6 mice with their respective controls.

Correlations between Markers of Disease Severity and the Pulmonary Immune Response to M. pneumoniae Respiratory Infection as Measured by BAL Cytokines and Chemokines
TNF-{alpha}, IFN-{gamma}, IL-6, IL-12, MIP-1{alpha}, and MCP-1 demonstrated a significant positive correlation with AO in M. pneumoniae–infected BALB/c mice 1 d after inoculation; IL-12 directly correlated with HPS (Table 3). BAL concentration of IL-12 demonstrated a significant positive correlation with M. pneumoniae quantitative BAL culture (r = 0.53, P = 0.016, n = 8) and IFN-{gamma} directly correlated with HPS (r = 0.70, P = 0.047, n = 8) in M. pneumoniae–infected C57Bl/6 mice 1 d after inoculation (Table 4).


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  		TABLE 3. Correlations of m. pneumoniae culture (log10cfu/ml), histopathologic score, and airway obstruction (penh values) with cytokine and chemokine concentrations in bronchoalveolar lavage samples (pg/ml) of balb/c mice 1 d after inoculation with m. pneumoniae

 

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  		TABLE 4. Correlations of m. pneumoniae culture (log10cfu/ml), histopathologic score, and airway obstruction (penh values) with cytokine and chemokine concentrations in bronchoalveolar lavage (pg/ml) of C57bl/6 mice 1 d after inoculation with m. pneumoniae

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The results presented characterize the host-dependent nature of acute M. pneumoniae respiratory infection. We inoculated BALB/c and C57BL/6 mice intranasally once with M. pneumoniae and evaluated groups of mice during the first week after inoculation. All mice became infected and displayed histologic inflammation. Notably, the BALB/c mice had worse pulmonary inflammation and greater peribronchial infiltrates than C57BL/6 mice at all time points evaluated. Most striking, however, were the differences found in pulmonary function. Only BALB/c mice developed significant airway obstruction compared with controls. Furthermore, after standardized, aerosolized methacholine challenge, BALB/c mice manifested significantly greater AHR compared with C57BL/6 mice, as well as BALB/c control mice. Thus, the BALB/c mice demonstrated a phenotype consistent with acute wheezing or asthma, compared with C57BL/6 mice. This emphasizes that the immunogenetic background (mouse strain) is important for the development of M. pneumoniae–associated AHR.

A variety of infectious disease models demonstrate variable host genetic susceptibility to disease expression (22, 23, 37–40). In such models, variation in susceptibility has frequently been correlated with differences in the patterns of immune responses. Therefore, the cytokine and chemokine concentrations in the BAL fluid of BALB/c and C57BL/6 mice during acute M. pneumoniae respiratory infection were measured. Each mouse strain had a distinct BAL cytokine and chemokine profile representing a host-dependent immune response to infection with M. pneumoniae. M. pneumoniae–infected BALB/c mice had significantly higher BAL concentrations of chemokines (KC and MIP-1{alpha}), proinflammatory cytokines (TNF-{alpha}, IL-1ß, and IL-6), and Th1 cytokines (IFN-{gamma} and IL-12), compared with infected C57BL/6 mice. Markers of disease severity correlated with the pulmonary immune response to M. pneumoniae as measured by BAL cytokines and chemokines. A strong positive correlation between ß-chemokines (MIP-1{alpha} and MCP-1), Th1 cytokines (IFN-{gamma} and IL-12), and pulmonary function abnormalities (AO) was found in BALB/c mice 1 d after inoculation with M. pneumoniae. No change in disease severity was observed with Th2 cytokines including IL-4 and IL-5. BALB/c mice did not demonstrate a Th2-biased immune response in this model of M. pneumoniae respiratory infection, which is consistent with previous studies (16, 18, 19). The expression pattern of IL-4, IL-5, IL-12p40, IFN-{gamma}, and TNF-{alpha} mRNA in the lung tissue as measured by real-time RT-PCR demonstrated a similar pattern to that seen in the BAL cytokine profile described above. Although the immune response at the site of infection differed between strains, the systemic response was similar.

Our previous investigations in M. pneumoniae–infected BALB/c mice have shown that the concentrations of TNF-{alpha}, IFN-{gamma}, IL-6, IL-12, KC, MIP-1{alpha}, and MCP-1 in BAL are significantly reduced by antibiotic therapy with clarithromycin, cethromycin, and azithromycin therapy (18–20). BAL concentrations of GM-CSF, IL-2, IL-4, and IL-10 were not affected by antibiotic therapy. In addition, the reductions observed in pulmonary cytokines and chemokines were associated with improvement in pulmonary cellular inflammation and airway obstruction in these studies. This also suggests that these cytokines and chemokines have a role in orchestrating pulmonary inflammation and function after M. pneumoniae infection.

In a similar model using the mouse pathogenic mycoplasma species M. pulmonis, it was demonstrated that certain mouse strains differ markedly in resistance to infection and progression of disease with C3H/HeN mice being more susceptible than C57BL/6 mice (41). The resistance of C57BL/6 mice to M. pulmonis is thought to be related to nonspecific, innate intrapulmonary host immunity which limits the extent of infection (41, 42). Cartner and coworkers have also investigated the response of various murine strains to infection with M. pulmonis by evaluating 17 inbred mouse strains, and found that resistance to murine mycoplasmosis is a complex trait controlled by multiple genes (43).

Previous studies evaluating the cytokine production in acute M. pulmonis respiratory infection showed that susceptible C3H mice had higher BAL concentrations of TNF-{alpha} and IL-6 and higher serum IL-6 concentrations when compared with resistant C57BL/6 mice (44). Evidence from an experimental model of reinfection with M. pneumoniae in mice indicate that the pulmonary inflammatory response is associated with an elevated expression of proinflammatory cytokines (TNF-{alpha}, IL-1ß, and IL-6) (45). These results are consistent with our findings and suggest that differences in production of proinflammatory cytokines could be among the factors explaining the variable host susceptibility to mycoplasma respiratory disease.

Although the host factors that control disease severity for any mycoplasma disease are not well defined, our results suggest that the higher production of pulmonary inflammatory and Th1 cytokines and chemokines in BALB/c mice compared with C57BL/6 mice is associated with more severe disease. An attractive hypothesis would be that the elevated chemokine concentrations found in the BAL of infected BALB/c mice were responsible for the greater peribronchial infiltrate found in these mice compared with C57BL/6 mice, coinciding with the significant increase in the BAL proinflammatory and Th1 cytokines. In addition, we speculate that the greater peribronchial infiltrate in BALB/c mice is at least in part responsible for the development of airway obstruction seen in BALB/c mice and not in C57BL/6 mice. Additional studies are needed to characterize the cellular nature of the peribronchial infiltrate and to evaluate the role of individual cytokines and chemokines in the pathogenesis of the AO and AHR associated with M. pneumoniae infection.

It has been shown that inbred mouse strains exhibit significant genetic variability in their susceptibility to develop AHR with allergen-sensitized C57BL/6 mice having attenuated AHR when compared with allergen-sensitized BALB/c mice and a number of other strains (46, 47). Based on the differences in expression of AHR, murine models of allergic asthma using different inbred mouse strains have provided insight into the major mechanisms that may result in allergic asthma (47, 48). In the present study, we demonstrated that M. pneumoniae respiratory infection can induce variable AO and AHR in a murine model without allergen presensitization.

Although we found that a distinct BAL cytokine and chemokine profile correlated with pulmonary function abnormalities when comparing M. pneumoniae infection in BALB/c mice and C57BL/6 mice, it is also known that other immunologic and genetic differences exist between these mouse strains, which could affect experimental outcomes. These differences include innate immunity (alveolar macrophages), mast cell tryptase, IL-9, total and specific IgE, and dendritic cell subsets (49–51). Furthermore, these two mouse strains have genetic differences in the H-2 region (murine major histocompatibility complex) and in other H-2–associated genes, mimicking the differences in major histocompatibility complex among humans (24, 29, 30).

It is worth mentioning that our present study evaluated M. pneumoniae respiratory infection using only one dose of the organism. Future studies need to determine the effects of larger and smaller infectious doses and even reinfection after adaptive immunity has developed.

In addition, there is ongoing controversy in the literature regarding the use of Penh as a measure of airway resistance in mice. Nonetheless, Penh as measured by whole-body unrestrained plethysmography has been previously validated in animal models of ovalbumin- and viral-induced AHR (33–36, 52). Previous studies in ovalbumin-sensitized and intranasally challenged BALB/c mice demonstrated that changes in airway hyperresponsiveness seen with whole-body unrestrained plethysmography were largely due to changes in the lung and not the nasopharynx (52). After intranasal inoculation with M. pneumoniae, it is possible that mice could also develop rhinitis; thus, the term "airway hyperresponsiveness" instead of "bronchial hyperresponsiveness" is used in the present study.

Asthma is a chronic inflammatory disease of the airways thought to be the result of complex interactions between the host's genetic background and diverse environmental factors (53, 54). Moreover, it is likely that the clinical syndrome of asthma is heterogeneous and multifactorial in nature (55). The traditional hypothesis for the pathogenesis of allergic asthma is based on a relative increase in Th2 cellular responses in combination with a decrease in Th1 responses. Recently, however, an increasingly complex inflammatory environment in asthma has been revealed, with several studies indicating that Th1 cell types and mediators may actually be enhanced in asthma (56, 57). Some Th1 processes may thus potentiate pathologic responses in individuals with asthma, in contrast to their traditional downregulatory role (58). Therefore, although Th2 cells play a critical role in the pathogenesis of asthma, the Th2 dysregulation hypothesis may not explain all the immunological processes that occur in asthma (59–61).

Respiratory tract infections with M. pneumoniae, C. pneumoniae, and respiratory viruses may influence asthma pathogenesis in distinct ways. Infection with M. pneumoniae has been associated with multiple aspects of asthma, including asthma initiation, exacerbation of asthma, and asthma chronicity. Wheezing in particular has been documented in 20–40% of children with acute M. pneumoniae infection. Although the pathogenesis of wheezing induced by infectious agents is not well defined, it is likely influenced by multiple factors including age, sex, environmental exposures, and genetic predisposition.

In summary, we have characterized a murine model of host-dependent M. pneumoniae lower respiratory tract infection with variable disease expression including AO, AHR, and histologic inflammation associated with the host immune response. The data presented are likely relevant to human M. pneumoniae infection, since humans, like mice, exhibit heterogeneity in susceptibility to M. pneumoniae respiratory disease, as well as other respiratory tract infections.


   Acknowledgments
 
The authors thank Naveed Ahmad for his assistance in statistical analysis.


   Footnotes
 
This work was supported by the KO8 NIH grant of R.D.H.

Conflict of Interest Statement: M.F-A. has no declared conflicts of interest; A.M.R. has no declared conflicts of interest; A.M. has no declared conflicts of interest; S.C-B. has no declared conflicts of interest; K.K. has no declared conflicts of interest; A.M.G. has no declared conflicts of interest; G.H.M. has no declared conflicts of interest; and R.D.H. has no declared conflicts of interest.

Received in original form June 18, 2004

Received in final form November 18, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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