Biomarkers of early-stage Mycoplasma pneumoniae pneumonia

Biomarkers of early-stage Mycoplasma pneumoniae pneumonia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biomarkers of early-stage Mycoplasma pneumoniae pneumonia Lulu Fang, Shaohu Huo, Zhenyu Ding, Wenhong Li, Wenli Li, Kang Lin, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3866039/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Early recognition of Mycoplasma pneumoniae infection and the severity of M. pneumoniae pneumonia (MPP) are difficult to ascertain because early signs of infection are atypical, with no obvious clinical manifestations or imaging characteristics. The inability to diagnosis early-stage MPP delays treatment and increases risks of progression to refractory MPP or severe pneumonia. Methods Here, we used a mouse model of MPP to investigate whether levels of S100 proteins or inflammatory factors in serum and bronchoalveolar lavage fluid (BALF) could be useful biomarkers of M. pneumoniae infection or MPP severity. The contents of S100A8, S100A9, Interleukin (IL)-6, and TNF-α in serum and BALF obtained from M. pneumoniae-infected mice were measure using enzyme-linked immunosorbent assays. Hematoxylin-eosin staining used to judge the severity of MPP showed lung tissue with obvious lesions. TUNEL staining indicated apoptosis in lung tissue of M. pneumoniae-infected mice. Results The serum levels of S100A8 in the high-dose group were higher on days 3 and 5 than those in the low-dose group. The serum levels of S100A9 in the infection group were higher on days 1 and 3 than those in the control group. Serum levels of TNF-α and IL-6 in the M. pneumoniae -infected groups than those in the control group. S100A8/A9 levels in BALF derived from mice receiving the high dose of M. pneumoniae were significantly higher than those in the control group.The BALF levels of TNF-α in the high-dose group were higher on days 1 and 3 than those in the control group.The levels of IL-6 in the high-dose group were higher than those in the control group and those in the low-dose group. The degree of apoptosis in both high- and low-dose groups was higher than that in the control groups, the degree of apoptosis in the high-dose group was higher on day 3 than that in the low-dose group. Conclusion These finding suggest that serum and BALF S100A8/A9 and TNF-α levels may be useful for early diagnosis of MPP and for differentiating MPP severity. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Mycoplasma pneumoniae is a highly contagious and common human pathogen that is easily spread through droplets and close contact[1]. M. pneumoniae infections occur frequently, and this bacterium is an important pathogen in community-acquired pneumonia[2, 3]. The symptoms of M. pneumoniae pneumonia (MPP) are atypical, showing no obvious clinical manifestations or imaging characteristics[4], making diagnosis difficult and delaying treatment[5, 6], and thus increasing the risk of progression to refractory MPP or severe pneumonia[7, 8]. The methods used to diagnose M. pneumoniae infection include serological testing and nucleic acid detection[9–11]. Detection of antibodies against M. - pneumoniae has limited value for early clinical diagnosis because these antibody tests have low sensitivity and specificity, and antibodies may not increase markedly in the serum of children with imperfect immune function[12, 13]. Although nucleic acid diagnostic technology is the gold standard for detecting bacterial infections, it cannot differentiate reinfection or the various phases of infection. Thus, this method is also not useful for the diagnosis of early-stage MPP. Therefore, early diagnostic methods and the ability to judge disease severity in MPP require further attention and development. The binding of calcium-dependent cytokines S100A8 and S100A9 to form heterodimer complexes is important for inducing chemotactic infiltration of inflammatory cells and enhancing cytokine activity in a variety of physiological and pathological processes[12,14–18]. S100A8 and S100A9 have more rapid and obvious reactions to inflammatory changes than the heparin-binding protein procalcitonin[19, 20]. Therefore, serum levels of S100A8/A9 may be useful for evaluating inflammation activity, acting as a risk indicator for infection. Thus, investigating changes in proteins and cytokines in serum or bronchoalveolar fluid (BALF) may lead to the identification of new biomarkers for MPP, enabling early diagnosis and differentiating the severity of disease. In the present study, we used a mouse model of MPP to assess the severity of MPP as assessed using a histopathological score, to detect cell apoptosis in pulmonary tissue, and to examine the levels of S100A8, S100A9, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in serum and BALF. Our results indicated that S100A8/A9 may be useful for early detection of M. pneumoniae infection and for assessing the severity of MPP. Materials and Methods M. pneumoniae culture and CCU assay A standard strain (FH, 15531) of M. pneumoniae was reconstituted in M. pneumoniae broth (Cat. No. LA7340, Solarbio; Beijing, China) at 37°C in 5% CO2 incubators. M. pneumoniae were serially diluted by tenfold in culture flasks containing LA7340 media. In the CCU assay, the CCU corresponds to the inverse of the dilution factor for a stable color change [12]. M. pneumoniae was cultured at concentrations of approximately 10 3 and 10 6 CCU/mL. The original stock was aliquoted and stored at − 80°C. Mouse model of MPP Male BALB/c mice (ages 6–8 weeks) were purchased from the Laboratory Animal Center of Anhui Medical University and adapted to the environment for 7 days before the start of the experiment. Mice were randomly allocated to the control group, M. pneumoniae high-dose (103 CCU/mL) group, and M.- pneumoniae low-dose (10 6 CCU/mL) group, three mice per group. Mice were infected by endotracheal intubation with 20 mL of M. pneumoniae at a dose of 10 3 CCU/mL or 10 6 CCU/mL, once per day for 2 days. The control group was given an equal volume of medium. The infection was carried out in the Biosafety Secondary Laboratory at the University of Science and Technology of China (Registration No. 20010033). All mice were free access to food and water. And all experimental procedures and protocols were conducted in accordance with the guidelines of the local animal care and use committee. Animal welfare and experimental design were approved by the Ethics Committee of Anhui Medical University (protocol code: LLSC20211519; approved on 6 January 2022).The study is reported in accordance with ARRIVE guidelines. PCR detection of M. pneumoniae DNA The animals were sacrificed by cervical dislocation after pentobarbital anesthesia to collect BALF and serum 1, 3, and 5 days after the second (final) M.- pneumoniae infection. Serum was obtained by centrifugation at 2000×g for 20 min. Left lungs were irrigated with 300 µL of saline three times to obtain BALF, which was preserved on ice. The BALF (50 µL) extracted from the lungs was cultured in M. pneumoniae medium for 7 days, and then 0.2 mL was obtained for PCR analysis. M. pneumoniae DNA was extracted using a SteadyPure Bacteria Genomic DNA Extraction Kit (Cat. No. AG21007, Accurate Biotechnology; Hunan, China) and a gel imaging system was used to detect a fragment of 453 bp. The M. pneumoniae DNA fragment was amplified using the following system: DNA template (9.5 µL), M. pneumoniae PCR reaction mix (12.5 µL total that also contained the specific forward primer 5’-CCAAGTGGATCCCTGATCTCTTTGGCGAC-3’ [1 µL], reverse primer 5’-GCAAATTGAATTCCCCGTTGTTCAGGATCAG-3’ [1 µL], and RNA free water [1 µL]). The PCR reaction was carried out under the following conditions: 30 cycles of 95°C for 1 min, 62°C for 1 min, and 72°C for 1 min. All experiments were conducted according to the manufacturer’s protocols. ELISA detection of S100 proteins and proinflammatory cytokines The levels of S100 proteins S100A8 and S100A9 and two of the most common and typical pro-inflammatory cytokines (IL-6 and TNF-α) were assessed for their inflammation responses in BALB/c mice infected with M. pneumoniae. In brief, serum and BALF samples were assessed for cytokine levels using mouse TNF-α ELISA Kits (Cat. No. ml002095-J, Shanghai Enzyme Biotechnology Co, Ltd; Shanghai, China) and mouse IL-6 ELISA Kits (Cat. No. ml002093-J, Enzy-linked Biotechnology) according to the manufacturer's instructions. Similarly, the levels of S100A8, S100A9, and IL-6 were detected using mouse S100A8 ELISA Kits (Cat. No. ml037986-J, Enzy-linked Biotechnology) and S100A9 ELISA Kits (Cat. No. ml037984-J, Enzy-linked Biotechnology). All specimens were stored at − 80°C before analysis. H&E and TUNEL staining of lung tissue Lung tissues from mice infected with M. pneumoniae and control mice were extracted and subjected to H&E staining, histopathological examination, and immunohistochemistry. The freshly resected lung tissues were fixed in 10% neutral-buffered formalin overnight and then embedded in paraffin. Tissue sections approximately 5µm thick were sliced and stained with H&E for light microscopic examination. The histopathological score of lung specimens includes the percentage of involved sites with peribronchiolar and bronchial infiltrates, quality of infiltrates, luminal exudates, perivascular infiltrates, and parenchymal pneumonia. Apoptosis staining were performed using TUNEL Apoptosis Detection Kits (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's instructions. The sections were mounted on glass slides, deparaffinized, treated with 20 µg/mL of proteinase K (Be-yotime Institute of Biotechnology) at 37°C for 20 min, and then washed in 1× Tris buffer for microscopic examination. TUNEL analysis was conducted using the image analysis software Image Pro Plus. Statistical Analysis Analyses were conducted using GraphPad Prism 5.0 software. All values are reported as means ± standard errors. All data were analyzed using repeated-measures two-way analysis of variance. Two-tailed P values < 0.05 were considered statistically significant. Results Concentration of M. pneumoniae in culture and induction of a mouse model of MPP To measure the concentration of M. pneumoniae in culture, we used the color-changing unit (CCU) assay. The shift in color was monitored across tenfold serially diluted solutions of M. pneumoniae , ranging from 10 1 to 10 10 CCU/mL. The highest dilution before the color shift of the medium was considered the CCU. As shown in Fig. 1 , the CCU was at a concentration of 10 6 CCU/mL M. pneumoniae . A mouse model of MPP was induced by endotracheal intubation of BALB/c mice with M. pneumoniae at concentrations of 10 3 CCU/mL and 10 6 CCU/mL. On day 1 after the final infection with M.- pneumoniae , the mice showed no respiratory signs of pneumonia. On days 3 and 5, their fur appeared unkempt, their eyes were closed, they were unresponsiveness to external stimuli, and they had poor appetite and mental status. In contrast, mice in the control group appeared normal. These manifestations indicated the successful establishment of an animal model of MPP. PCR analysis of M. pneumoniae in BALF To confirm that mice were successfully infected with M. pneumoniae , we collected BALF from their lungs and extracted M. pneumoniae DNA by polymerase chain reaction (PCR) after culturing the BALF in medium for 7 days. BALF from healthy mice served as a control. Figure 2 shows the results of M.- pneumoniae DNA bands displayed on a gel imaging system. A fragment of M. pneumoniae DNA was detected as 453 base pairs (bp) in length. Bands of the 453-bp fragment were observed in BALF collected from both the high-dose (10 6 CCU/mL) and low-dose (10 3 CCU/mL) groups. The intensity of the band at 453 bp in the high-dose group was brighter than that in the low-dose group. In the high-dose group, the intensity of the bands on days 1 and 3 after infection were brighter than those on day 5, but there was no significant difference in band intensity across days in the low-dose group. Thus, it was demonstrated that mycoplasma successfully infected mice and most pronounced in the first three days. All groups of gels under the same exposure conditions. The full-length blots are presented in Supplementary Fig. 1. Levels of S100 proteins and proinflammatory cytokines in serum To assess whether two S100 proteins (S100A8 and S100A9) and two representative pro-inflammatory cytokines (IL-6 and TNF-α) were highly expressed after M. pneumoniae infection, we collected serum to determine their levels by using enzyme-linked immunosorbent assays (ELISAs). The results showed that serum levels of S100A8 in the high-dose group on days 1, 3, and 5 were higher than those in the control group. In the low-dose group, serum levels of S100A8 were higher on days 1 and 5 than those in the control group (Fig. 3 A). In addition, the serum levels of S100A8 in the high-dose group were higher on days 3 and 5 than those in the low-dose group (Fig. 3 A). The serum levels of S100A9 in the high-dose group on days 1, 3 and 5 were higher than those in the control group. Serum levels of S100A9 in the low-dose group were higher on days 1 and 3 than those in the control group (Fig. 3 B). There was no significant difference in S100A9 serum levels between the high- and low-dose groups (Fig. 3 B). Serum levels of TNF-α in the M. pneumoniae -infected groups were higher on days 1, 3 and 5 than those in the control group, with serum levels of TNF-α in the high-dose group higher than those in the low-dose group on days 1 and 3 (Fig. 3 C). Serum levels of IL-6 in the M. pneumoniae -infected groups were higher on days 3 and 5 than those in the control group. There was no significant difference in IL-6 serum levels between the high- and low-dose groups (Fig. 3 D). Taken together, these results indicated that infection with M. pneumoniae significantly increased the serum S100 protein and cytokine levels assessed herein and that the increases appeared to be dose dependent for S100A8 and TNF-α. Levels of S100A8, S100A9, TNF-α, and IL-6 in BALF The levels of S100A8, S100A9, IL-6, and TNF-α in BALF derived from mice were analyzed by ELISA (Fig. 4 ). The results showed that S100A8 levels in BALF derived from mice receiving the high dose of M. pneumoniae were significantly higher on days 1, 3, and 5 than those in the control group (Fig. 4 A). BALF levels of S100A8 in the low-dose group were higher on day 1 than those in the control group. In addition, the levels of S100A8 in the high-dose group were higher on days 3 and 5 than those in the low dose group. The BALF levels of S100A9 in the high-dose group were significantly higher than those in the control group; the BALF levels of S100A9 in the low-dose group were higher on day 3 than those in the control group (Fig. 4 B). Moreover, the BALF levels of S100A9 in the high-dose group were higher on day 5 than those in the low-dose group. The BALF levels of TNF-α in the high-dose group were higher on days 1 and 3 than those in the control group (Fig. 4 C). There was no significant difference in BALF levels of TNF-α between the low-dose group and the control group and no significant difference between the high-dose and low-dose groups. The levels of IL-6 in the high-dose group were higher on days 3 and 5 after M. pneumoniae infection than those in the low-dose group and the control group (Fig. 4 D). Taken together, these results indicated that infection with M. pneumoniae significantly increased the S100 protein and cytokine levels assessed here in BALF and that the increases appeared to be dose dependent for S100A8/9 and IL-6. Morphological changes in lung tissue of mice after infection with M. pneumoniae As shown in Fig. 5 , compared with healthy mice, mice infected with M. pneumoniae showed obvious lesions to lung tissue, manifested as inflammatory infiltration, congestion, and pulmonary edema. On day 3, mice infected with a high dose of M. pneumoniae displayed numerous patchy inflammatory infiltrations and severe pulmonary edema on both the right and left lungs, whereas mice infected with a low dose of M. pneumoniae displayed inflammatory lesions only on one side. These lesions progressively worsened over time, which was consistent with the changes in levels of S100 proteins and proinflammatory cytokines. Hematoxylin and eosin (H&E) staining and histological evaluation Lungs were collected from M. pneumoniae –infected mice and control mice on days 1, 3, and 5. H&E staining was used to evaluate the histological changes in lung tissue after the infection. As shown in Fig. 6 A, alveolar septa were thickened, alveolar cavities were narrowed, and numerous inflammatory cells (neutrophils, lymphocytes, and macrophages) infiltrated the lungs of mice from the high-dose group compared with both the low-dose and control groups. A histopathological scoring system was used to assess the pathology of the pulmonary infection, with higher scores indicating worse pathology. As shown in Fig. 6 B, the histopathological scores in the M. pneumoniae –infected groups were significantly higher than those of the control group and were highest on day 3 after infection. There was no significant difference in histopathological scores between the high-dose group and the low-dose group. These results indicated that lung tissue damage in this mouse model of MPP was dependent on both concentration of M. pneumoniae and time after infection, with the most severe damage on the third day after infection. Apoptosis assessed by TUNEL assay To further evaluate lung tissue damage, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to detect apoptosis in lung tissue caused by infection with M. pneumoniae . Under a light microscope, normal cells appeared blue, and apoptotic cells appeared brown. As shown in Fig. 7 A, apoptotic cells were widely distributed around bronchi, alveolar spaces, and alveolar septa in M. pneumoniae –infected lung tissue, especially in the high-dose group. Compared with that in control mice, the mean integral optical density of the apoptotic cells in infected mice was significantly increased (* P < 0.001). The degree of apoptosis in both high- and low-dose groups was higher on days 1, 3, and 5 after infection than that in the control groups (Fig. 7 B). In addition, the degree of apoptosis in the high-dose group was higher on day 3 than that in the low-dose group. These findings indicated that infection with M. pneumoniae significantly increased the apoptosis in lung tissue and that the increases appeared to be dose dependent. Discussion 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. Declarations Ethics Approval All experimental procedures and protocols were conducted in accordance with the guidelines of the local animal care and use committee. Animal welfare and experimental design were approved by the Ethics Committee of Anhui Medical University (protocol code: LLSC20211519; approved on 6 January 2022). The study is reported in accordance with ARRIVE guidelines. Consent to Participate Not applicable. Consent for Publication Not applicable (animal study). Conflicts of Interest The authors declare no conflict of interest. Funding This research was funded by Anhui Province Key Research and Development Program Project, grant number 9021364202. Author Contribution S.D., Y.Z., L.W., and L.F. conceived and designed the experiments, L.F., S.H., and Z.D., W.L., W.L. performed the experiments, L.F., K.L., and B.S. analyzed the data, L. F., S.H., and Z.D. wrote the paper. All authors reviewed the manuscript. Acknowledgements We would like to express our gratitude to all those who have helped us during Data Availability Statement All data generated or analyzed during this study are included in this published article and are available from the corresponding author upon request. References Sanchez-Vargas FM, Gomez-Duarte OG. Mycoplasma pneumoniae-an emerging extra-pulmonary pathogen. Clin Microbiol Infect. 2008;14(2):105–17. Shah SS. Mycoplasma pneumoniae as a Cause of Community-Acquired Pneumonia in Children. Clin Infect Dis. 2019;68(1):13–4. Izumikawa K. 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Respir Res, 2021. 22(1). Zhao J, Li YY, Zhang W. The clinical significance of IL-6 s and IL-27 s in Bronchoalveolar lavage fluids from children with mycoplasma pneumoniae pneumonia. BMC Infect Dis, 2020. 20(1). Ding Y, et al. High expression of HMGB1 in children with refractory Mycoplasma pneumoniae pneumonia. BMC Infect Dis. 2018;18(1):439. Li G et al. High co-expression of TNF-alpha and CARDS toxin is a good predictor for refractory Mycoplasma pneumoniae pneumonia. Mol Med, 2019. 25(1). Fan HF, et al. Distribution and Expression of IL-17 and Related Cytokines in Children with Mycoplasma pneumoniae Pneumonia. Jpn J Infect Dis. 2019;72(6):387–93. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3866039","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":268740029,"identity":"3e6bbde0-335f-411e-a2cc-07cb19519605","order_by":0,"name":"Lulu Fang","email":"","orcid":"","institution":"the First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lulu","middleName":"","lastName":"Fang","suffix":""},{"id":268740030,"identity":"ceef5a41-515f-4605-9683-f112890b242f","order_by":1,"name":"Shaohu Huo","email":"","orcid":"","institution":"the First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shaohu","middleName":"","lastName":"Huo","suffix":""},{"id":268740031,"identity":"7b6466e7-c7c0-4a4e-a532-5a7150cc3dd9","order_by":2,"name":"Zhenyu Ding","email":"","orcid":"","institution":"Wannan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Ding","suffix":""},{"id":268740032,"identity":"6250c33c-3614-4dd6-97ba-3e4f2a0afdab","order_by":3,"name":"Wenhong Li","email":"","orcid":"","institution":"the First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenhong","middleName":"","lastName":"Li","suffix":""},{"id":268740033,"identity":"aed72063-4db5-4463-ad65-ae0437c63614","order_by":4,"name":"Wenli Li","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenli","middleName":"","lastName":"Li","suffix":""},{"id":268740034,"identity":"dc79534a-1e99-4d6a-9f04-28bc295de23a","order_by":5,"name":"Kang Lin","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kang","middleName":"","lastName":"Lin","suffix":""},{"id":268740035,"identity":"9522f5ab-08a8-4837-86f0-4c2c55fd4fa7","order_by":6,"name":"Bing Shen","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Shen","suffix":""},{"id":268740036,"identity":"6c95e7e2-6bee-4211-b839-a80c1216e8e6","order_by":7,"name":"Linding Wang","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Linding","middleName":"","lastName":"Wang","suffix":""},{"id":268740037,"identity":"9f95c861-9c5f-42ad-a17d-d1b36a2f6033","order_by":8,"name":"Yulin Zhu","email":"","orcid":"","institution":"the First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yulin","middleName":"","lastName":"Zhu","suffix":""},{"id":268740038,"identity":"0d9ecf35-d96f-4254-9bdd-a7f5449361cf","order_by":9,"name":"Ding shengang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDCCw2DShsEATLMRryWNFC0HoBqJ18J3nPfwyx+/zsubi50xYPhQdpiBf3YDfi2Sh/nSrHn7bhvunJ1jwDjj3GEGiTsH8GsxOMxjZszYc5txw+0cA2beNqALJRIIazH82XPOHqzlL5FajB/w/DiQCNbCSIwWSaAtzLwNyckbbqcVHOw5l84jcYOAFr7zZ4w//vhjZ7vhdvLGBz/KrOX4ZxDQAgRsEoxtENYBIOYhqB4ImD8w/CFG3SgYBaNgFIxYAABwa0dQB3XEOAAAAABJRU5ErkJggg==","orcid":"","institution":"the First Affiliated Hospital of Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ding","middleName":"","lastName":"shengang","suffix":""}],"badges":[],"createdAt":"2024-01-15 09:35:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3866039/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3866039/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50174550,"identity":"85e6597c-fa29-45f6-8736-b2fabe375906","added_by":"auto","created_at":"2024-01-25 16:12:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":281015,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the color-changing assay for \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e. The concentration of \u003cem\u003eM.- pneumoniae\u003c/em\u003e in tube 6 is 10\u003csup\u003e6\u003c/sup\u003e CCU/mL.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3866039/v1/66e356e71096afaa1a56d89e.png"},{"id":50174549,"identity":"f53d4c97-0c98-4a2e-a0d1-b66fac5c78d9","added_by":"auto","created_at":"2024-01-25 16:12:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30901,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e DNA in mouse bronchoalveolar fluid after infection. The intensity of the band for the fragment at 453 bp represents the level of \u003cem\u003eM. pneumoniae\u003c/em\u003e in the initial bacterial fluid (A) that infected mice and each group on day 1 (B), day 3 (C) and day 5 (D) after infection.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3866039/v1/21e76026547ad65f5618dcf9.png"},{"id":50174554,"identity":"ae73af53-eee4-4bf4-9459-1f0d9dbf1d5d","added_by":"auto","created_at":"2024-01-25 16:12:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27951,"visible":true,"origin":"","legend":"\u003cp\u003eSerum levels of S100A8 (A), S100A9 (B), TNF-α (C), and of IL-6 (D) in mice infected with a high or low dose of \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e, analyzed by ELISA on days 1, 3, and 5 after infection. Values represent the mean ± SEM; n = 3; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 vs. control; #P \u0026lt; 0.05, ##P \u0026lt; 0.01 vs. low dose.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3866039/v1/9418b970a57452ee1d6e87de.png"},{"id":50174551,"identity":"635463e7-01bd-4fa6-8179-6a71f9fad21f","added_by":"auto","created_at":"2024-01-25 16:12:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":27934,"visible":true,"origin":"","legend":"\u003cp\u003eBronchoalveolar fluid levels of S100A8 (A), S100A9 (B), TNF-α (C), and of IL-6 (D) in mice infected with a high or low dose of \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e, analyzed by ELISA on days 1, 3, and 5 after infection. Values represent the mean ± SEM; n = 3; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 vs. control; #P \u0026lt; 0.05, ##P \u0026lt; 0.01 vs. low dose.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3866039/v1/183bc487b6a49ecf77fddae4.png"},{"id":50174553,"identity":"ded13ca1-60d3-470f-b72b-f1df3eb78f58","added_by":"auto","created_at":"2024-01-25 16:12:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":705014,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of the gross lung specimens from control mice and mice infected with high or low doses of \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e on 1, 3, and 5 days after infection. Black circles indicate tissue congestion and inflammatory cell infiltration; yellow arrows indicate tissue edema.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3866039/v1/ebe8d37c2aab9d29092b1eb9.png"},{"id":50174555,"identity":"ba157938-ed08-4dac-874a-b3c7a4abd767","added_by":"auto","created_at":"2024-01-25 16:12:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":861865,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin and eosin (H\u0026amp;E) staining and histopathological scores for lung tissue derived from mice infected with \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e. (A) Representative images of H\u0026amp;E-stained lung tissue sections on days 1, 3, and 5 after infection. (B) Histopathological scores of lung tissue damage in mice infected with a high dose or low dose of M. pneumoniae and control groups. Scale bar, 50 µm. Values represent the mean ± SEM; n = 3; **P \u0026lt; 0.01, ***P \u0026lt; 0.001 vs. control.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3866039/v1/a04f09438dc11498b99bc3d5.png"},{"id":50175106,"identity":"a3ec5a21-db43-4a6a-8b1f-bebf2c6de666","added_by":"auto","created_at":"2024-01-25 16:20:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":848221,"visible":true,"origin":"","legend":"\u003cp\u003eCell apoptosis in mouse lung tissue and histopathological scores after infection with \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e. (A) Representative images of TUNEL staining on days 1, 3, and 5 after infection. Apoptotic cells appear as brown granules (red arrows), and normal cells as blue granules. Scale bar, 100 µm. (B) Mean integrated optical density (IOD) of high-dose, low-dose and control groups based on TUNEL staining. Values represent the mean ± SEM; n = 3; **P \u0026lt; 0.01, ***P \u0026lt; 0.001 vs. control, ##P \u0026lt; 0.01 vs. low dose.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3866039/v1/7d9a13d2fd12c6172c5c6de2.png"},{"id":69957653,"identity":"fcdd26a7-38eb-4af1-85ae-774eadb5e7a1","added_by":"auto","created_at":"2024-11-27 03:46:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4234075,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3866039/v1/eb3dad79-e444-4d0f-95f9-1f2a2bb91c1f.pdf"},{"id":50174556,"identity":"3d1d2e88-840b-4001-a6f0-47ac8e29c9ef","added_by":"auto","created_at":"2024-01-25 16:12:15","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":123701,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3866039/v1/07f1ae09925604d0a62b161d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biomarkers of early-stage Mycoplasma pneumoniae pneumonia","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e is a highly contagious and common human pathogen that is easily spread through droplets and close contact[1]. \u003cem\u003eM. pneumoniae\u003c/em\u003e infections occur frequently, and this bacterium is an important pathogen in community-acquired pneumonia[2, 3]. The symptoms of \u003cem\u003eM. pneumoniae\u003c/em\u003e pneumonia (MPP) are atypical, showing no obvious clinical manifestations or imaging characteristics[4], making diagnosis difficult and delaying treatment[5, 6], and thus increasing the risk of progression to refractory MPP or severe pneumonia[7, 8]. The methods used to diagnose \u003cem\u003eM. pneumoniae\u003c/em\u003e infection include serological testing and nucleic acid detection[9\u0026ndash;11]. Detection of antibodies against \u003cem\u003eM.\u003c/em\u003e-\u003cem\u003epneumoniae\u003c/em\u003e has limited value for early clinical diagnosis because these antibody tests have low sensitivity and specificity, and antibodies may not increase markedly in the serum of children with imperfect immune function[12, 13]. Although nucleic acid diagnostic technology is the gold standard for detecting bacterial infections, it cannot differentiate reinfection or the various phases of infection. Thus, this method is also not useful for the diagnosis of early-stage MPP. Therefore, early diagnostic methods and the ability to judge disease severity in MPP require further attention and development.\u003c/p\u003e \u003cp\u003eThe binding of calcium-dependent cytokines S100A8 and S100A9 to form heterodimer complexes is important for inducing chemotactic infiltration of inflammatory cells and enhancing cytokine activity in a variety of physiological and pathological processes[12,14\u0026ndash;18]. S100A8 and S100A9 have more rapid and obvious reactions to inflammatory changes than the heparin-binding protein procalcitonin[19, 20]. Therefore, serum levels of S100A8/A9 may be useful for evaluating inflammation activity, acting as a risk indicator for infection. Thus, investigating changes in proteins and cytokines in serum or bronchoalveolar fluid (BALF) may lead to the identification of new biomarkers for MPP, enabling early diagnosis and differentiating the severity of disease.\u003c/p\u003e \u003cp\u003eIn the present study, we used a mouse model of MPP to assess the severity of MPP as assessed using a histopathological score, to detect cell apoptosis in pulmonary tissue, and to examine the levels of S100A8, S100A9, interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) in serum and BALF. Our results indicated that S100A8/A9 may be useful for early detection of \u003cem\u003eM. pneumoniae\u003c/em\u003e infection and for assessing the severity of MPP.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eM. pneumoniae culture and CCU assay\u003c/h2\u003e \u003cp\u003eA standard strain (FH, 15531) of \u003cem\u003eM. pneumoniae\u003c/em\u003e was reconstituted in \u003cem\u003eM. pneumoniae\u003c/em\u003e broth (Cat. No. LA7340, Solarbio; Beijing, China) at 37\u0026deg;C in 5% CO2 incubators. \u003cem\u003eM. pneumoniae\u003c/em\u003e were serially diluted by tenfold in culture flasks containing LA7340 media. In the CCU assay, the CCU corresponds to the inverse of the dilution factor for a stable color change [12]. \u003cem\u003eM. pneumoniae\u003c/em\u003e was cultured at concentrations of approximately 10\u003csup\u003e3\u003c/sup\u003e and 10\u003csup\u003e6\u003c/sup\u003e CCU/mL. The original stock was aliquoted and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMouse model of MPP\u003c/h2\u003e \u003cp\u003eMale BALB/c mice (ages 6\u0026ndash;8 weeks) were purchased from the Laboratory Animal Center of Anhui Medical University and adapted to the environment for 7 days before the start of the experiment. Mice were randomly allocated to the control group, \u003cem\u003eM. pneumoniae\u003c/em\u003e high-dose (103 CCU/mL) group, and \u003cem\u003eM.- pneumoniae\u003c/em\u003e low-dose (10\u003csup\u003e6\u003c/sup\u003e CCU/mL) group, three mice per group. Mice were infected by endotracheal intubation with 20 mL of \u003cem\u003eM. pneumoniae\u003c/em\u003e at a dose of 10\u003csup\u003e3\u003c/sup\u003e CCU/mL or 10\u003csup\u003e6\u003c/sup\u003e CCU/mL, once per day for 2 days. The control group was given an equal volume of medium. The infection was carried out in the Biosafety Secondary Laboratory at the University of Science and Technology of China (Registration No. 20010033). All mice were free access to food and water. And all experimental procedures and protocols were conducted in accordance with the guidelines of the local animal care and use committee. Animal welfare and experimental design were approved by the Ethics Committee of Anhui Medical University (protocol code: LLSC20211519; approved on 6 January 2022).The study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePCR detection of M. pneumoniae DNA\u003c/h2\u003e \u003cp\u003eThe animals were sacrificed by cervical dislocation after pentobarbital anesthesia to collect BALF and serum 1, 3, and 5 days after the second (final) \u003cem\u003eM.- pneumoniae\u003c/em\u003e infection. Serum was obtained by centrifugation at 2000\u0026times;g for 20 min. Left lungs were irrigated with 300 \u0026micro;L of saline three times to obtain BALF, which was preserved on ice. The BALF (50 \u0026micro;L) extracted from the lungs was cultured in \u003cem\u003eM. pneumoniae\u003c/em\u003e medium for 7 days, and then 0.2 mL was obtained for PCR analysis. M. pneumoniae DNA was extracted using a SteadyPure Bacteria Genomic DNA Extraction Kit (Cat. No. AG21007, Accurate Biotechnology; Hunan, China) and a gel imaging system was used to detect a fragment of 453 bp. The \u003cem\u003eM. pneumoniae\u003c/em\u003e DNA fragment was amplified using the following system: DNA template (9.5 \u0026micro;L), \u003cem\u003eM. pneumoniae\u003c/em\u003e PCR reaction mix (12.5 \u0026micro;L total that also contained the specific forward primer 5\u0026rsquo;-CCAAGTGGATCCCTGATCTCTTTGGCGAC-3\u0026rsquo; [1 \u0026micro;L], reverse primer 5\u0026rsquo;-GCAAATTGAATTCCCCGTTGTTCAGGATCAG-3\u0026rsquo; [1 \u0026micro;L], and RNA free water [1 \u0026micro;L]). The PCR reaction was carried out under the following conditions: 30 cycles of 95\u0026deg;C for 1 min, 62\u0026deg;C for 1 min, and 72\u0026deg;C for 1 min. All experiments were conducted according to the manufacturer\u0026rsquo;s protocols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eELISA detection of S100 proteins and proinflammatory cytokines\u003c/h2\u003e \u003cp\u003eThe levels of S100 proteins S100A8 and S100A9 and two of the most common and typical pro-inflammatory cytokines (IL-6 and TNF-α) were assessed for their inflammation responses in BALB/c mice infected with M. pneumoniae. In brief, serum and BALF samples were assessed for cytokine levels using mouse TNF-α ELISA Kits (Cat. No. ml002095-J, Shanghai Enzyme Biotechnology Co, Ltd; Shanghai, China) and mouse IL-6 ELISA Kits (Cat. No. ml002093-J, Enzy-linked Biotechnology) according to the manufacturer's instructions. Similarly, the levels of S100A8, S100A9, and IL-6 were detected using mouse S100A8 ELISA Kits (Cat. No. ml037986-J, Enzy-linked Biotechnology) and S100A9 ELISA Kits (Cat. No. ml037984-J, Enzy-linked Biotechnology). All specimens were stored at \u0026minus;\u0026thinsp;80\u0026deg;C before analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eH\u0026amp;E and TUNEL staining of lung tissue\u003c/h2\u003e \u003cp\u003eLung tissues from mice infected with \u003cem\u003eM. pneumoniae\u003c/em\u003e and control mice were extracted and subjected to H\u0026amp;E staining, histopathological examination, and immunohistochemistry. The freshly resected lung tissues were fixed in 10% neutral-buffered formalin overnight and then embedded in paraffin. Tissue sections approximately 5\u0026micro;m thick were sliced and stained with H\u0026amp;E for light microscopic examination. The histopathological score of lung specimens includes the percentage of involved sites with peribronchiolar and bronchial infiltrates, quality of infiltrates, luminal exudates, perivascular infiltrates, and parenchymal pneumonia. Apoptosis staining were performed using TUNEL Apoptosis Detection Kits (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's instructions. The sections were mounted on glass slides, deparaffinized, treated with 20 \u0026micro;g/mL of proteinase K (Be-yotime Institute of Biotechnology) at 37\u0026deg;C for 20 min, and then washed in 1\u0026times; Tris buffer for microscopic examination. TUNEL analysis was conducted using the image analysis software Image Pro Plus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAnalyses were conducted using GraphPad Prism 5.0 software. All values are reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors. All data were analyzed using repeated-measures two-way analysis of variance. Two-tailed P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eConcentration of M. pneumoniae in culture and induction of a mouse model of MPP\u003c/h2\u003e \u003cp\u003eTo measure the concentration of M. pneumoniae in culture, we used the color-changing unit (CCU) assay. The shift in color was monitored across tenfold serially diluted solutions of \u003cem\u003eM. pneumoniae\u003c/em\u003e, ranging from 10\u003csup\u003e1\u003c/sup\u003e to 10\u003csup\u003e10\u003c/sup\u003e CCU/mL. The highest dilution before the color shift of the medium was considered the CCU. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the CCU was at a concentration of 10\u003csup\u003e6\u003c/sup\u003e CCU/mL \u003cem\u003eM. pneumoniae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA mouse model of MPP was induced by endotracheal intubation of BALB/c mice with \u003cem\u003eM. pneumoniae\u003c/em\u003e at concentrations of 10\u003csup\u003e3\u003c/sup\u003e CCU/mL and 10\u003csup\u003e6\u003c/sup\u003e CCU/mL. On day 1 after the final infection with \u003cem\u003eM.- pneumoniae\u003c/em\u003e, the mice showed no respiratory signs of pneumonia. On days 3 and 5, their fur appeared unkempt, their eyes were closed, they were unresponsiveness to external stimuli, and they had poor appetite and mental status. In contrast, mice in the control group appeared normal. These manifestations indicated the successful establishment of an animal model of MPP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePCR analysis of M. pneumoniae in BALF\u003c/h2\u003e \u003cp\u003eTo confirm that mice were successfully infected with \u003cem\u003eM. pneumoniae\u003c/em\u003e, we collected BALF from their lungs and extracted \u003cem\u003eM. pneumoniae\u003c/em\u003e DNA by polymerase chain reaction (PCR) after culturing the BALF in medium for 7 days. BALF from healthy mice served as a control. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the results of \u003cem\u003eM.- pneumoniae\u003c/em\u003e DNA bands displayed on a gel imaging system. A fragment of \u003cem\u003eM. pneumoniae\u003c/em\u003e DNA was detected as 453 base pairs (bp) in length. Bands of the 453-bp fragment were observed in BALF collected from both the high-dose (10\u003csup\u003e6\u003c/sup\u003e CCU/mL) and low-dose (10\u003csup\u003e3\u003c/sup\u003e CCU/mL) groups. The intensity of the band at 453 bp in the high-dose group was brighter than that in the low-dose group. In the high-dose group, the intensity of the bands on days 1 and 3 after infection were brighter than those on day 5, but there was no significant difference in band intensity across days in the low-dose group. Thus, it was demonstrated that mycoplasma successfully infected mice and most pronounced in the first three days. All groups of gels under the same exposure conditions. The full-length blots are presented in Supplementary Fig.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLevels of S100 proteins and proinflammatory cytokines in serum\u003c/h2\u003e \u003cp\u003eTo assess whether two S100 proteins (S100A8 and S100A9) and two representative pro-inflammatory cytokines (IL-6 and TNF-α) were highly expressed after \u003cem\u003eM. pneumoniae\u003c/em\u003e infection, we collected serum to determine their levels by using enzyme-linked immunosorbent assays (ELISAs). The results showed that serum levels of S100A8 in the high-dose group on days 1, 3, and 5 were higher than those in the control group. In the low-dose group, serum levels of S100A8 were higher on days 1 and 5 than those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In addition, the serum levels of S100A8 in the high-dose group were higher on days 3 and 5 than those in the low-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The serum levels of S100A9 in the high-dose group on days 1, 3 and 5 were higher than those in the control group. Serum levels of S100A9 in the low-dose group were higher on days 1 and 3 than those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). There was no significant difference in S100A9 serum levels between the high- and low-dose groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Serum levels of TNF-α in the \u003cem\u003eM. pneumoniae\u003c/em\u003e-infected groups were higher on days 1, 3 and 5 than those in the control group, with serum levels of TNF-α in the high-dose group higher than those in the low-dose group on days 1 and 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Serum levels of IL-6 in the \u003cem\u003eM. pneumoniae\u003c/em\u003e-infected groups were higher on days 3 and 5 than those in the control group. There was no significant difference in IL-6 serum levels between the high- and low-dose groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Taken together, these results indicated that infection with \u003cem\u003eM. pneumoniae\u003c/em\u003e significantly increased the serum S100 protein and cytokine levels assessed herein and that the increases appeared to be dose dependent for S100A8 and TNF-α.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLevels of S100A8, S100A9, TNF-α, and IL-6 in BALF\u003c/h2\u003e \u003cp\u003eThe levels of S100A8, S100A9, IL-6, and TNF-α in BALF derived from mice were analyzed by ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The results showed that S100A8 levels in BALF derived from mice receiving the high dose of \u003cem\u003eM. pneumoniae\u003c/em\u003e were significantly higher on days 1, 3, and 5 than those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). BALF levels of S100A8 in the low-dose group were higher on day 1 than those in the control group. In addition, the levels of S100A8 in the high-dose group were higher on days 3 and 5 than those in the low dose group. The BALF levels of S100A9 in the high-dose group were significantly higher than those in the control group; the BALF levels of S100A9 in the low-dose group were higher on day 3 than those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Moreover, the BALF levels of S100A9 in the high-dose group were higher on day 5 than those in the low-dose group. The BALF levels of TNF-α in the high-dose group were higher on days 1 and 3 than those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). There was no significant difference in BALF levels of TNF-α between the low-dose group and the control group and no significant difference between the high-dose and low-dose groups. The levels of IL-6 in the high-dose group were higher on days 3 and 5 after \u003cem\u003eM. pneumoniae\u003c/em\u003e infection than those in the low-dose group and the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Taken together, these results indicated that infection with \u003cem\u003eM. pneumoniae\u003c/em\u003e significantly increased the S100 protein and cytokine levels assessed here in BALF and that the increases appeared to be dose dependent for S100A8/9 and IL-6.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMorphological changes in lung tissue of mice after infection with M. pneumoniae\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, compared with healthy mice, mice infected with \u003cem\u003eM. pneumoniae\u003c/em\u003e showed obvious lesions to lung tissue, manifested as inflammatory infiltration, congestion, and pulmonary edema. On day 3, mice infected with a high dose of \u003cem\u003eM. pneumoniae\u003c/em\u003e displayed numerous patchy inflammatory infiltrations and severe pulmonary edema on both the right and left lungs, whereas mice infected with a low dose of \u003cem\u003eM. pneumoniae\u003c/em\u003e displayed inflammatory lesions only on one side. These lesions progressively worsened over time, which was consistent with the changes in levels of S100 proteins and proinflammatory cytokines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin and eosin (H\u0026amp;E) staining and histological evaluation\u003c/h2\u003e \u003cp\u003eLungs were collected from \u003cem\u003eM. pneumoniae\u003c/em\u003e–infected mice and control mice on days 1, 3, and 5. H\u0026amp;E staining was used to evaluate the histological changes in lung tissue after the infection. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, alveolar septa were thickened, alveolar cavities were narrowed, and numerous inflammatory cells (neutrophils, lymphocytes, and macrophages) infiltrated the lungs of mice from the high-dose group compared with both the low-dose and control groups. A histopathological scoring system was used to assess the pathology of the pulmonary infection, with higher scores indicating worse pathology. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, the histopathological scores in the \u003cem\u003eM. pneumoniae\u003c/em\u003e–infected groups were significantly higher than those of the control group and were highest on day 3 after infection. There was no significant difference in histopathological scores between the high-dose group and the low-dose group. These results indicated that lung tissue damage in this mouse model of MPP was dependent on both concentration of \u003cem\u003eM. pneumoniae\u003c/em\u003e and time after infection, with the most severe damage on the third day after infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis assessed by TUNEL assay\u003c/h2\u003e \u003cp\u003eTo further evaluate lung tissue damage, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to detect apoptosis in lung tissue caused by infection with \u003cem\u003eM. pneumoniae\u003c/em\u003e. Under a light microscope, normal cells appeared blue, and apoptotic cells appeared brown. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, apoptotic cells were widely distributed around bronchi, alveolar spaces, and alveolar septa in \u003cem\u003eM. pneumoniae\u003c/em\u003e–infected lung tissue, especially in the high-dose group. Compared with that in control mice, the mean integral optical density of the apoptotic cells in infected mice was significantly increased (* P \u0026lt; 0.001). The degree of apoptosis in both high- and low-dose groups was higher on days 1, 3, and 5 after infection than that in the control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In addition, the degree of apoptosis in the high-dose group was higher on day 3 than that in the low-dose group. These findings indicated that infection with \u003cem\u003eM. pneumoniae\u003c/em\u003e significantly increased the apoptosis in lung tissue and that the increases appeared to be dose dependent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eM. pneumoniae\u003c/em\u003e 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 \u003cem\u003eM. pneumoniae\u003c/em\u003e 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 \u003cem\u003eM. pneumoniae\u003c/em\u003e–infected mice; this increase was greater in mice infected with a high dose. (3) \u003cem\u003eM. pneumoniae\u003c/em\u003e 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 \u003cem\u003eM. pneumoniae\u003c/em\u003e–infected mice were consistent with the severity of MPP manifestations. Taken together, these results indicated that \u003cem\u003eM. pneumoniae\u003c/em\u003e 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.\u003c/p\u003e\u003cp\u003eThe 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 \u003cem\u003eM. pneumoniae\u003c/em\u003e infection in mice, and inflammatory cell infiltration was found in their lung tissue, especially in mice infected with the high dose of \u003cem\u003eM. pneumoniae\u003c/em\u003e. These results suggested that \u003cem\u003eM. pneumoniae\u003c/em\u003e 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 \u003cem\u003eM. pneumoniae\u003c/em\u003e–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.\u003c/p\u003e\u003cp\u003eProinflammatory cytokines are important components in the inflammatory response. IL-6 and TNF-α play important roles in predicting \u003cem\u003eM. pneumoniae\u003c/em\u003e 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 \u003cem\u003eM. pneumoniae\u003c/em\u003e 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 \u003cem\u003eM. pneumoniae\u003c/em\u003e were consistent with the dose of \u003cem\u003eM. pneumoniae\u003c/em\u003e. The higher the concentration of \u003cem\u003eM. pneumoniae\u003c/em\u003e 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 \u003cem\u003eM.- pneumoniae\u003c/em\u003e infection and may not be useful in differentiating MPP severity.\u003c/p\u003e\u003cp\u003eIn conclusion, the levels of S100A8/A9 and TNF-α were high in the serum and BALF of mice with \u003cem\u003eM.- pneumoniae\u003c/em\u003e 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics Approval\u003c/strong\u003e \u003cp\u003eAll experimental procedures and protocols were conducted in accordance with the guidelines of the local animal care and use committee. Animal welfare and experimental design were approved by the Ethics Committee of Anhui Medical University (protocol code: LLSC20211519; approved on 6 January 2022). The study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for Publication\u003c/strong\u003e \u003cp\u003eNot applicable (animal study).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflicts of Interest\u003c/strong\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by Anhui Province Key Research and Development Program Project, grant number 9021364202.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.D., Y.Z., L.W., and L.F. conceived and designed the experiments, L.F., S.H., and Z.D., W.L., W.L. performed the experiments, L.F., K.L., and B.S. analyzed the data, L. F., S.H., and Z.D. wrote the paper. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe would like to express our gratitude to all those who have helped us during\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed during this study are included in this published article and are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSanchez-Vargas FM, Gomez-Duarte OG. Mycoplasma pneumoniae-an emerging extra-pulmonary pathogen. Clin Microbiol Infect. 2008;14(2):105\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah SS. Mycoplasma pneumoniae as a Cause of Community-Acquired Pneumonia in Children. Clin Infect Dis. 2019;68(1):13\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIzumikawa K. 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Int J Biochem Cell Biol. 2001;33(7):637\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchiopu A, Cotoi OS. S100A8 and S100A9: DAMPs at the Crossroads between Innate Immunity, Traditional Risk Factors, and Cardiovascular Disease. Mediators of Inflammation, 2013. 2013.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing Z et al. Targeting S100A9 Reduces Neutrophil Recruitment, Inflammation and Lung Damage in Abdominal Sepsis. Int J Mol Sci, 2021. 22(23).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChakraborty D, et al. Alarmin S100A8 Activates Alveolar Epithelial Cells in the Context of Acute Lung Injury in a TLR4-Dependent Manner. Front Immunol. 2017;8:1493.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao BY et al. S100A9 blockade prevents lipopolysaccharide-induced lung injury via suppressing the NLRP3 pathway. Respir Res, 2021. 22(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao J, Li YY, Zhang W. The clinical significance of IL-6 s and IL-27 s in Bronchoalveolar lavage fluids from children with mycoplasma pneumoniae pneumonia. BMC Infect Dis, 2020. 20(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing Y, et al. High expression of HMGB1 in children with refractory Mycoplasma pneumoniae pneumonia. BMC Infect Dis. 2018;18(1):439.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi G et al. High co-expression of TNF-alpha and CARDS toxin is a good predictor for refractory Mycoplasma pneumoniae pneumonia. Mol Med, 2019. 25(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan HF, et al. Distribution and Expression of IL-17 and Related Cytokines in Children with Mycoplasma pneumoniae Pneumonia. Jpn J Infect Dis. 2019;72(6):387\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3866039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3866039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEarly recognition of Mycoplasma pneumoniae infection and the severity of M. pneumoniae pneumonia (MPP) are difficult to ascertain because early signs of infection are atypical, with no obvious clinical manifestations or imaging characteristics. The inability to diagnosis early-stage MPP delays treatment and increases risks of progression to refractory MPP or severe pneumonia.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHere, we used a mouse model of MPP to investigate whether levels of S100 proteins or inflammatory factors in serum and bronchoalveolar lavage fluid (BALF) could be useful biomarkers of M. pneumoniae infection or MPP severity. The contents of S100A8, S100A9, Interleukin (IL)-6, and TNF-α in serum and BALF obtained from M. pneumoniae-infected mice were measure using enzyme-linked immunosorbent assays. Hematoxylin-eosin staining used to judge the severity of MPP showed lung tissue with obvious lesions. TUNEL staining indicated apoptosis in lung tissue of M. pneumoniae-infected mice.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe serum levels of S100A8 in the high-dose group were higher on days 3 and 5 than those in the low-dose group. The serum levels of S100A9 in the infection group were higher on days 1 and 3 than those in the control group. Serum levels of TNF-α and IL-6 in the \u003cem\u003eM. pneumoniae\u003c/em\u003e-infected groups than those in the control group. S100A8/A9 levels in BALF derived from mice receiving the high dose of \u003cem\u003eM. pneumoniae\u003c/em\u003e were significantly higher than those in the control group.The BALF levels of TNF-α in the high-dose group were higher on days 1 and 3 than those in the control group.The levels of IL-6 in the high-dose group were higher than those in the control group and those in the low-dose group. The degree of apoptosis in both high- and low-dose groups was higher than that in the control groups, the degree of apoptosis in the high-dose group was higher on day 3 than that in the low-dose group.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese finding suggest that serum and BALF S100A8/A9 and TNF-α levels may be useful for early diagnosis of MPP and for differentiating MPP severity.\u003c/p\u003e","manuscriptTitle":"Biomarkers of early-stage Mycoplasma pneumoniae pneumonia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-25 16:12:10","doi":"10.21203/rs.3.rs-3866039/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d041889c-07e4-4cc2-a2bb-16ba2620cca9","owner":[],"postedDate":"January 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-27T03:38:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-25 16:12:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3866039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3866039","identity":"rs-3866039","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}