This study represents the first analysis of the interaction of M. pneumoniae
CARDS toxin with the immune system of an experimental animal model. While many bacterial pathogens produce toxins that modify the host response to infection, the recently discovered CARDS toxin, in contrast to many bacterial toxins, is unique in that it is both a vacuolating and ADP-ribosylating toxin. Currently, it is not clear how ADP-ribosylation or the phenotype of vacuolation correlates with the ability of the CARDS toxin to induce inflammation. However, the data presented in this study indicate that CARDS toxin is capable of inducing pro-inflammatory cytokine expression in the lungs of mice and baboons. In mice treated with a single dose of rCARDS toxin, the toxin induces a transient pro-inflammatory response characterized by increased expression of IL-1, 6, 12p40, 17, and TNF-α as well as a number of inflammatory chemokines. In the baboon, G-CSF, IL-1ra, 6, 8, MIP-1α, RANTES, and IFN-γ were readily detectable after rCARDS toxin treatment. The pattern of pro-inflammatory cytokine expression in both mice and baboons is remarkably similar to that observed during human pulmonary infection with M. pneumoniae 
, including the prolonged expression of IL-12p40 after rCARDS toxin exposure. The relatively high levels of IL-12p40 but lower levels of IL-12p70 may suggest a role for IL-23, but this remains to be investigated. Hardy and co-workers have demonstrated that mice lacking IL-12 infected with M. pneumoniae
have less severe disease and rapidly resolve inflammatory changes in lung histology 
. In contrast, treatment of mice with rIL-12 during the acute phase of infection prolongs acute disease, increases pro-inflammatory cytokine levels, and exacerbates inflammatory pathology as well as changes in airway function 
. rCARDS toxin appears to be capable of inducing several but not all of the cytokines reported to be produced after M. pneumoniae
infection, which is predictable given the complexity of mycoplasma antigens presented to the immune system during infection.
Clearly, exposure to rCARDS toxin not only increases pro-inflammatory cytokine levels but also leads to profound changes in inflammatory histopathology. In vitro the CARDS toxin is an ADP-ribosylating and vacuolating toxin that is capable of inducing vacuoles in both cell and organ cultures (27). Here we demonstrated that early after a single exposure to rCARDS toxin, frequent and consistent vacuolization of the bronchiolar epithelium in vivo occurs that resolves by day 4, which corroborates our initial studies in tissue and organ culture 
. Interestingly, the kinetics of the resolution of the vacuolization is consistent with rapid regeneration of bronchiolar epithelium.
The CARDS toxin-mediated inflammatory infiltrates are very similar to those observed during M. pneumoniae
infection, and the perivascular lesions suggest a hematological origin (). rCARDS toxin eventually induces a robust peribronchial inflammation evident starting at day 4 post exposure. How specific cytokines and chemokines actually contribute to the development of these inflammatory infiltrates remains to be identified, but previous studies suggest that IL-12 might play a significant role 
. The cellular infiltrates are unusual in that they are predominantly lymphocytic in nature and increase in severity over a seven-day period in a dose-dependent manner following a single exposure to toxin. The presence of lymphoblasts after toxin exposure is uncommon during the host response to most bacterial toxins, and it is unclear what the significance of this finding is. However, it indicates a complex and interdependent relationship between M. pneumoniae
CARDS toxin and the immune system. For example, the same lymphocytic aspect of the histopathology is observed following both M. pneumoniae
infection and intoxication, suggesting a role for toxin during infection.
Pathophysiologically, exposure to CARDS toxin and the associated inflammatory responses in the lungs could account for the changes observed in pulmonary function after infection. Mouse models of chronic respiratory infection with M. pneumoniae
have been established and have demonstrated that these chronically infected animals develop alteration in lung compliance, functional AO and AHR, as well as histological evidence for airway inflammation accompanied by fibrosis 
. Here we report that mice exposed to a single dose of rCARDS toxin develop statistically significant prolonged AO over 21 days and AHR at day 2-post exposure. The fact that AHR waned prior to resolution of AO indicates that AHR requires a sustained exposure to toxin, which would be typical of the infectious process. Prolonged AHR similar to what is observed during infection may also have been observed with a continuous toxin challenge. These data suggest that a single exposure to CARDS toxin is sufficient to cause significant changes in histopathology and airway function in mice that may be clinically relevant to a range of M. pneumoniae
has been implicated as a causative agent or an exacerbating factor in a number of acute and chronic airway-associated inflammatory conditions ranging from tracheobronchitis and community acquired pneumonia to asthma and COPD in both children and adults 
. In adults, M. pneumoniae
has been detected in the airways of chronic, stable asthmatics using PCR with significantly greater frequency than in non-asthmatic control subjects 
. In a randomized, double blind, placebo-controlled trial of prolonged (6 week) clarithromycin therapy in 55 adult subjects with chronic, stable asthma  M. pneumoniae
was detected by PCR in the airways of 23 of the 55 asthmatics. Prolonged clarithromycin therapy resulted in a significant improvement in pulmonary function (FEV1
) only in the PCR-positive asthmatics (p
0.05) and was without effect in the PCR-negative asthmatics (p
0.85), directly linking M. pneumoniae
and mycoplasma products to airway dysfunction. A conclusive causal link between M. pneumoniae
infection and reactive airway disease is lacking but based on the ability of rCARDS toxin to induce robust inflammation and both AO and AHR in mice, it would be reasonable to predict that CARDS toxin would elicit similar responses in humans. Further, the improvement in airway function after antibiotic treatment (42) suggests that a virulence factor sensitive to protein synthesis inhibitors might play a role in the pathogenesis of disease. CARDS toxin is a likely candidate based on the in vivo effects of rCARDS toxin reported in this study and the fact that the magnitude of CARDS toxin expression by M. pneumoniae
is markedly increased in vivo using the mouse model of infection relative to toxin expression during broth culture (T.R. Kannan et al Submitted).
Currently, there is no reliable way to estimate the total amount of CARDS toxin produced in vivo during M. pneumoniae infection. Therefore, it is difficult to make direct comparisons between treatment with rCARDS toxin and toxin produced during infection although the host response to rCARDS exposure or infection shares similarities (3,27). During infection many factors can lead to an inflammatory response, and it is unlikely that a single M. pneumoniae product accounts for the overall cytokine responses, the corresponding lymphocytic inflammation, as well as changes in airway function. During infection, the airway-associated histopathology and airway dysfunction, which we observed after rCARDS treatment alone, can be further amplified by other immuno-reactive mycoplasma products present at the site of infection. However, the results of this study suggest that the CARDS toxin is sufficient to induce major inflammatory phenotypes associated with M. pneumoniae infection in rodents and primates. These data support the concept that the CARDS toxin plays a fundamentally important role in the pathogenesis of M. pneumoniae infection and the sequelae associated with infection. Clinically, it is likely that CARDS toxin is a significant cause of human morbidity associated with M. pneumoniae infection.