M. pneumoniae is a pathogen commonly associated with community-acquired pneumonia in children[2]. Up to 18% of children with MPP require hospitalization, and the incidence of severe and refractory MPP is continually increasing[21]. Therefore, it is critical to have the proper clinical tools to evaluate the severity of MPP in a timely manner. However, sensitive indicators to diagnose MPP are still limited. In our previous study, we found that the levels of S100A8/A9 in the BALF and serum were significantly increased among children with MPP, and we speculated that these S100 proteins may be good biomarkers for diagnosis of MPP[22]. To further evaluate the value of S100A8/A9 in MPP diagnosis in the present study, we generated a mouse model of MPP. Our key findings were that (1) mice given a high dose of M. pneumoniae developed more pronounced congestion, edema, and inflammatory cell infiltration in their lungs than mice given a low-dose or control mice. (2) The levels of S100A8, S100A9, IL-6, and TNF-α in BALF and serum were significantly increased in M. pneumoniae–infected mice; this increase was greater in mice infected with a high dose. (3) M. pneumoniae infection caused apoptosis in pulmonary tissue, which was greater in mice infected with a high dose. (4) The significantly increased levels of S100A8/A9 in BALF and serum in M. pneumoniae–infected mice were consistent with the severity of MPP manifestations. Taken together, these results indicated that M. pneumoniae infection induced apoptosis of lung cells in a concentration-dependent manner and that S100A8 and S100A9 may be useful biomarkers to differentiate the severity of MPP, providing a potential new tool for the clinical diagnosis of MPP in children.
The two calcium-binding proteins S100A8 and S100A9 are abundant in the cytoplasm of neutrophils and mononuclear phagocytes[23, 24]. When the body is infected with bacteria or is injured, neutrophils and monocytes—the main components in the initial stage of acute inflammatory response—rapidly move to the infection site and secrete S100A8/A9, resulting in the increase of S100A8/A9 in the tissue at the early infection stage[25–27]. S100A9 levels in lung tissue and BALF have been reported to be elevated in response to lipopolysaccharide-induced lung injury, and S100A9 is considered an important inflammatory mediator contributing to the progression of lipopolysaccharide-induced lung injury[28]. In the present study, S100A8 and S100A9 levels in serum and BALF were increased after M. pneumoniae infection in mice, and inflammatory cell infiltration was found in their lung tissue, especially in mice infected with the high dose of M. pneumoniae. These results suggested that M. pneumoniae infection led to increased S100A8 and S100A9 concentrations in serum and BALF, and that S100A8 and S100A9 level changes may be of potential value in differentiating the severity of MPP. In addition, our results showed that cell apoptosis occurred in the lung tissue of M. pneumoniae–infected mice. This finding is consistent with a previous finding by our group that elevated S100A8/A9 causes alveolar epithelial cell apoptosis[22]. In summary, S100A8 and S100A9 may be involved in the development of MPP and thus may be inflammatory markers useful for differentiating the severity of MPP.
Proinflammatory cytokines are important components in the inflammatory response. IL-6 and TNF-α play important roles in predicting M. pneumoniae infection and differentiating the severity of MPP[29, 30]. Li et al. found that TNF-α and IL-6 levels in BALF of patients with refractory MPP were significantly higher than those of non-refractory MPP and that TNF-α has the potential to be used as a biomarker to distinguish refractory from non-refractory MPP[31]. Fan et al. found that systemic inflammation or local inflammation in lung tissue can be identified by an elevated level of TNF-α in BALF from children with MPP[32]. Our present study similarly showed that the levels of TNF-α and IL-6 in serum and BALF of mice infected with M. pneumoniae were increased, especially in the high-dose group. We also found that the TNF-α levels in serum beginning the first day after mice were infected with M. pneumoniae were consistent with the dose of M. pneumoniae. The higher the concentration of M. pneumoniae that mice were infected with, the more obvious the lesions were in lung tissue and the higher the TNF-α levels in serum were. Although no significant differences were observed in BALF TNF-α levels among the high- and low-dose or control groups, we noted that TNF-α levels were nonsignificantly higher in the high-dose group than in the low-dose group, which were nonsignificantly higher than those in the control group. This result suggests that the severity of MPP may be associated with increased TNF-α levels in both serum and BALF, which is consistent with the findings reported in previous studies[31]. The IL-6 levels in serum and BALF were not significantly increased the first day after infection and did not significantly increase in BALF in the low-dose group on days 3 or 5; however, IL-6 levels in both serum and BALF increased on days 3 and 5 after infection in the high-dose group. These findings suggested that IL-6 may not be as sensitive as S100A8/A9 and TNF-α in response to M.- pneumoniae infection and may not be useful in differentiating MPP severity.
In conclusion, the levels of S100A8/A9 and TNF-α were high in the serum and BALF of mice with M.- pneumoniae infection and were significantly higher in mice with more severe infection. These findings suggest new tools for use in the early clinical diagnosis of MPP, in the prediction of the development of MPP, and in the differentiation of MPP severity.