Analysis of Pulmonary Inflammation and Function in the Mouse and Baboon after Exposure to Mycoplasma pneumoniae CARDS Toxin

Mycoplasma pneumoniae produces an ADP-ribosylating and vacuolating toxin known as the CARDS (Community Acquired Respiratory Distress Syndrome) toxin that has been shown to be cytotoxic to mammalian cells in tissue and organ culture. In this study we tested the ability of recombinant CARDS (rCARDS) toxin to elicit changes within the pulmonary compartment in both mice and baboons. Animals responded to a respiratory exposure to rCARDS toxin in a dose and activity-dependent manner by increasing the expression of the pro-inflammatory cytokines IL-1α, 1β, 6, 12, 17, TNF-α and IFN-γ. There was also a dose-dependent increase in several growth factors and chemokines following toxin exposure including KC, IL-8, RANTES, and G-CSF. Increased expression of IFN-γ was observed only in the baboon; otherwise, mice and baboons responded to CARDS toxin in a very similar manner. Introduction of rCARDS toxin to the airways of mice or baboons resulted in a cellular inflammatory response characterized by a dose-dependent early vacuolization and cytotoxicity of the bronchiolar epithelium followed by a robust peribronchial and perivascular lymphocytic infiltration. In mice, rCARDS toxin caused airway hyper-reactivity two days after toxin exposure as well as prolonged airway obstruction. The changes in airway function, cytokine expression, and cellular inflammation correlate temporally and are consistent with what has been reported for M. pneumoniae infection. Altogether, these data suggest that the CARDS toxin interacts extensively with the pulmonary compartment and that the CARDS toxin is sufficient to cause prolonged inflammatory responses and airway dysfunction

Delayed Inflammatory Response to Primary Pneumonic Plague Occurs in Both Outbred and Inbred Mice

Yersinia pestis is the causative agent of plague, a disease that can manifest as either bubonic or pneumonic plague. An interesting feature of plague is that it is a rapidly progressive disease, suggesting that Y. pestis either evades and/or suppresses the innate immune response to infection. Therefore, the early host response during the course of primary pneumonic plague was investigated in two mouse strains, the outbred strain CD1 and the inbred strain C57BL/6. A comparative analysis of the course of disease in these two strains of mice indicated that they are susceptible to intranasal Y. pestis CO92 infection and have similar 50% lethal doses and kinetics of infection with respect to colonization of the lung, liver, and spleen. Significantly, in both strains of mice, robust neutrophil recruitment to the lungs was not observed until 48 h after infection, suggesting that there was a delay in inflammatory cell recruitment to the site of infection. In addition, proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha, gamma interferon, IL-12p70, monocyte chemoattractant protein 1) and chemokines (KC, MIP-2) in the bronchoalveolar lavage fluids were not readily detected until 48 h after infection, which coincided with the increase in polymorphonuclear leukocyte (PMN) recruitment to the lungs. In comparison, CD1 mice with gram-negative pneumonia caused by Klebsiella pneumoniae exhibited strong inflammatory responses early in infection, with PMNs comprising the majority of the cells in the bronchoalveolar lavage fluid 24 h postinfection, indicating that PMN recruitment to the lungs could occur earlier in this infection than in Y. pestis infection. Together, our results indicate that there is a delay in the recruitment of neutrophils to the lungs in the mouse model of primary plague pneumonia that correlates with delayed expression of proinflammatory cytokines and chemokines in both outbred and inbred mice

Mouse perivascular lesions evident in the lung 4 days after <i>M. pneumoniae</i> infection.

<p>A) Low power micrograph shows scattered alveolar exudates (circle) proximal to a venule (v) with a mural cellular infiltrate of small mononuclear cells, predominately small lymphocytes. B) is a 4-fold magnification of Panel A) showing that the vessel wall is infiltrated with multilayered small lymphocytes. The endothelium is edematous, and adherent inflammatory cells are occasionally evident (arrow). Compare this histopathology to similar lesions induced by active rCARDS toxin presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007562#pone-0007562-g005" target="_blank">Figure 5A & B</a>. Hematoxylin and eosin, original magnifications 10× and 40× respectively.</p

Vacuolization of bronchiolar epithelium after CARDS toxin exposure.

<p>Two days after IN exposure to 43.7 pmol of active rCARDS toxin or HI toxin, histological changes in lung specimens were evaluated. A) Bronchiolar epithelial cells appear normal in the HI toxin–treated lung. B) Extensive vacuolization of the airway epithelium is evident in rCARDS toxin-treated lung. B = bronchiole. Hematoxylin and eosin, X 40 & 20 original magnification, respectively.</p

Quantifiable inflammatory histopathology observed in rCARDS toxin-treated mice.

<p>Histopathology was scored in a blinded fashion, and sections were given a numerical score as described in the Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007562#s2" target="_blank">Methods</a>. The most significant changes were observed in animals treated with 700 pmol of rCARDS on days 4 and 7 post-treatment. Data represents the average ± standard deviation with 8 to10 mice/time point/treatment group. Data sets were analyzed for statistical significance by comparing animals treated with the indicated amount of toxin to the corresponding group of animals treated with HI toxin * = p<0.05, ** = p<0.005.</p