Tofacitinib treats Mycoplasma Pneumoniae Pneumonia by inhibiting the CXCR4 molecule on the CD4+ T lymphocytes

Tofacitinib treats Mycoplasma Pneumoniae Pneumonia by inhibiting the CXCR4 molecule on the CD4+ T lymphocytes | 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 Tofacitinib treats Mycoplasma Pneumoniae Pneumonia by inhibiting the CXCR4 molecule on the CD4 + T lymphocytes Kaiwen Wang, Li Guo, Yongqi Zhang, Haiting Yang, Hui Du, Jiangfeng Zhao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6018592/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 Objective: To compare the differences in CXCR4 expression on the surface of CD4 + T lymphocytes between children with severe Mycoplasma pneumoniae pneumonia (SMPP) and those with common Mycoplasma pneumoniae pneumonia (Non-SMPP) and verify in vitro the potential application value of Tofacitinib as an adjuvant therapy for MPP by affecting the CXCR4 signaling pathway. Methods: This study recruited 267 children with Mycoplasma pneumoniae pneumonia (MPP), including 42 cases of SMPP and 225 cases of Non-SMPP at Jiading District Central Hospital in Shanghai from 2023 to 2024. Laboratory tests conducted within 24 hours after admission were used as baseline data, and the differences in CD4 + CXCR4 + expression between SMPP and Non-SMPP patients were compared. Animal experiments with different medicines were performed to evaluate the mechanism and therapeutic value of Tofacitinib as an adjuvant therapy for MPP. Results: Compared with Non-SMPP, children with SMPP had more fever (p=0.0062), longer durations of hospital stays (p<0.0001), higher level of erythrocyte sedimentation rate (p=0.0161) and especial the proportion of CD4 + CXCR4 + T lymphocytes (p<0.0001).However, there were no significant differences in C-reactive protein, procalcitonin and lactate dehydrogenase levels between SMPP and Non-SMPP group. In animal experiments, compared to wild-type mice, the levels of inflammatory cytokines (IL-6, IL-8, IP-10, and IL-2) were significantly elevated in the bronchoalveolar lavage fluid (BALF) of MPP mice. Notably, the sole ligand of CXCR4, CXCL12 was also markedly increased in the BALF of MPP mice. Tofacitinib could significantly reduce the expression of CXCR4 on the surface of CD4 + cells in MPP mice, as well as decrease the expression of a series of inflammatory cytokines. Conclusion: In summary, the proportion of CD4 + CXCR4 + T cells may be served as a predictive indicator of clinical severity for the MPP. Tofacitinib can reduce the release of inflammatory cytokines by inhibiting the CXCL12/CXCR4 signaling pathway and may be used as an adjuvant therapy for MPP. Mycoplasma pneumoniae pneumonia CD4+CXCR4+ lymphocyte Cytokine Tofacitinib Therapy Figures Figure 1 Figure 2 Figure 3 Introduction Mycoplasma pneumoniae pneumonia (MPP) is a common type of community-acquired pneumonia that mainly occurs in children aged 5 and above in China [ 1 ] . The symptoms of MPP are varied, including fever, cough, sore throat, etc., and are also related to acute exacerbation of asthma. Some with worsening symptoms and imaging findings are defined as severe MPP (SMPP), which may lead to serious intrapulmonary and extrapulmonary complications, such as lung abscesses, bronchiolitis obliterans, bronchiectasis, pulmonary embolism, myocarditis and encephalitis [ 2 , 3 ] . With the lifting of non-pharmaceutical interventions against COVID-19, the infection rate of Mycoplasma pneumoniae (MP) in children and the number of hospitalizations related to MP increased sharply in 2023 [ 4 ] . The clinical manifestations of the affected children were heterogeneous. Although most children were mild and self-limiting, severe, and refractory cases were also on the rise, accounting for up to 15% according to the latest reports [ 5 ] . Early diagnosis, accurate evaluation and effective treatment are of great significance in reducing the incidence and sequelae of SMPP. The diagnosis of MPP is not difficult, but there is a lack of potential biomarkers for clinical assessment of the disease condition and phenotype. Currently, commonly used indicators such as blood leukocytes, C-reactive protein (CRP), lactate dehydrogenase (LDH), ferritin, or certain cytokines are not highly specific or sensitive [ 6 ] . The pathogenesis of MPP mainly involves direct damage caused by MP and abnormal immune response of the host to MP, with the immune activation of T lymphocytes being considered as a crucial factor. Additionally, studies have shown that in the lungs of MPP children with pneumonia as the primary symptom, there is significant infiltration of Mycoplasma-specific Th1 and Th2 cells, which is mainly driven by chemokines and their receptors [ 7 ] . Another research suggested that CXCR4 may play a role in regulating the migration of inflammatory cells in MPP. When MP infects the respiratory tract, CXCL12 (the ligand of CXCR4) is upregulated at the site of infection, attracting CXCR4 positive inflammatory cells to migrate to the infected area [ 8 ] . These cells participate in immune responses and pathogen clearance by releasing inflammatory mediators and cytokines. However, the expression and specific mechanisms of CXCR4 in MPP are not fully elucidated. This study further explored the expression significance and regulatory mechanisms of CXCR4 in MPP and preliminarily investigated the therapeutic potential of Tofacitinib targeting CXCR4 in MPP mice. Methods Patients A total of 267 MPP children who underwent physical examination were prospectively enrolled in the pediatric department of Jiading District Central Hospital from November 2023 to May 2024. The inclusion criteria for MPP children are: (1) Children aged 1-12 years with pneumonia shown on chest imaging and meeting the diagnostic criteria for community-acquired pneumonia (CAP); (2) Positive MP nucleic acid test (collected from nasopharyngeal aspirates) or a fourfold or more increase in serum anti-MP IgM titer during the non-acute phase. The exclusion criteria are: (1) Children with heart disease, congenital immune deficiency, HIV-positive status, or malignant tumors; (2) The co-presence of other pathogens in the child's body fluids or blood detected by culture, or viral antigen test, or interferon-γ release assays, etc.; (3) Children who have a history of taking immunosuppressants, bronchodilators, or leukotriene receptor antagonists within 2 weeks prior to admission. The study was approved by Ethics Committee of Ren Ji Hospital Jiading Branch, Shanghai Jiao Tong University School of Medicine (Ethics number:2024K05). The cultivation of Mycoplasma pneumoniae Weigh out 30.8 g of ATCC Medium: 2611 (Shandong Top-Bioengineering Co., Ltd.，China) and add it to 780 ml of deionized water. After heating to dissolve, aliquot the solution. Then, perform autoclaving at 121°C for 15 minutes. Allow it to cool below 50°C. In a sterile environment, add 50 ml of CMRL-1066 tissue culture medium (Amylet Scientific, China) that has been sterilized by filtration, as well as 170 ml of fetal bovine serum (Gibco, USA) that has been inactivated at 55°C for 1 hour. Mix well, aliquot, and set aside for future use. The Mycoplasma pneumoniae strain (ATCC ® 15531 TM , Type Culture Collection, USA) was diluted with 0.5 mL PPLO broth (Pleuropneumonia-Like Organisms, BD Difco™ 211414) and inoculated into liquid culture medium. The culture was incubated at 37°C under 5% CO₂ for 24-72 hours. Subculturing was performed when turbidity was observed in the liquid medium. The bacterial concentration was determined using the Color Changing Units (CCU) assay before subsequent experimental use. Animal Model and Treatment C57BL/6 mice weighing 20-25 g (Shanghai Lab. Animal Research Center, China) were anesthetized by intraperitoneal injection of 3 mg phenobarbital sodium (Sangon Biotech (Shanghai) Co., Ltd, China). 0.2 ml of Mycoplasma pneumoniae liquid (ATCC ® 15531 TM , Type Culture Collection, USA) at logarithmic growth phase with 1 × 10 6 color changing units (ccu)/ml was dripped into the nasal cavity. After 14 days, most of the mice developed respiratory symptoms, and were pathologically confirmed to have pneumonia. An osmotic minipump (Alza Pharmaceuticals, Palo Alto, CA, USA) containing 200 μl saline (respectively with 3 mg/kg/day AMD3100, Sigma-Aldrich, USA; 6 mg/kg/day Tofacitinib, MedChemExpress, China; 20 mg/kg/day Azithromycin, MedChemExpress, China) was implanted subcutaneously into the left suprascapular region through an incision at the base of neck. According to the manufacturer's guidelines, AMD3100 was administered continuously from day 1 to day 14, Tofacitinib from day 3 to day 14, and Azithromycin from day 3 to day 7. As a control experiment, a minipump containing only saline was also used. Flow cytometry Blood was collected from the peripheral vein of children with MPP or the retro-orbital sinus of mice (on the 14th day after treatment) and then stained with FIFC-CD4, PerCP-CD3 and APC-CXCR4 (BD Biosciences- Pharmingen, San Diego, California, USA). The CXCR4 expression on the surface of CD4 + lymphocytes was measured using Cellquest software on the FACS Calibur flow cytometer (BD Biosciences-Pharmingen, San Diego, California, USA). HE staining The lungs of mice (on the 14th day after treatment) were fixed with 0.5 ml of 10% formalin (MACKLIN, China) and then embedded in paraffin. The paraffin blocks were cut into 3-4 μm sections, dewaxed and dried. The slides were stained in hematoxylin (MACKLIN, China) for 8 min, followed by rinsing and immersion in eosin solution (MACKLIN, China) for 4 min. Afterwards, the slides were dehydrated and cleared by immersing in alcohol of different concentrations and xylene sequentially. Finally, the slides were mounted with neutral balsam and observed under a microscope to assess the lung structure. Multiple fluorescence immunohistochemistry (TSA technology) After removing the blocking solution from the slice, rabbit anti-mouse CD4 antibody (1:1500, abcam, United Kingdom) and rabbit anti-mouse CXCR4 antibody (1:300, abcam, United Kingdom) were incubated in a humid chamber at 37°C for 2 hours. Subsequently, HRP-Goat Anti-Rabbit IgG H&L (abcam, 1:200) was incubated in the dark at room temperature for 30 min, and then TSA working solution (GEPbio, Shanghai Guipu Biotechnology Co., Ltd., China) was probed for 10 min. The panoramic digital scanning system (KFbio, Ningbo Jiangfeng Biotechnology Information Co., Ltd.，China) were used for analysis after incubating with DAPI staining solution for 10 min. Bronchoalveolar lavage and cell classification The distal end of the mouse trachea was ligated, and an intravenous indwelling needle was inserted into the trachea. After connecting the syringe, the trachea was gently lavaged with 1 ml saline for 5 times and followed by collecting the lavage fluid 3 times. After centrifugation at 1200 rpm for 5 min, the precipitate was resuspended in 1 ml liquid for cell counting, and then stained with Diff-Quick (Baxter, Miami, USA) for cell classification. Measurement of Mycoplasma DNA in BALF MP-DNA was performed using the FQ-PCR kit (Da'an Gene Co., Ltd., China). Specimen processing steps are as follows: i. Four volumes of saline were added to the bronchoalveolar lavage fluid (BALF), which was then repeatedly pipetted with a 1 ml pipette tip and left in 4°C overnight to ensure complete liquefaction of the BALF; ii. After mixing, 1 ml of the mixture was aspirated into a 1.5 ml tube and centrifuged at 12,000 rpm for 5 min; iii. Discarded the supernatant and add 50 μl DNA extraction buffer, which was thoroughly mixed and subjected to constant temperature treatment at 100°C for 10 min; iv. The mixture was centrifuged at 12,000 rpm for 5 min and prepared for use. The PCR amplification and data processing were conducted according to the manufacturer's instructions. using the PEGeneAmp 5700 system (ABI, USA). Analysis of cytokines and chemokines in BALF 24 inflammatory factors in mice were analyzed with the MILLIPLEX® Mouse Cytokine/Chemokine Magnetic Bead kit (Millipore, USA), following the instructions strictly and using Bio-Plex® Manager™ Software 6.0 (Bio-Plex, USA) for analysis. Statistical analysis All data analysis were performed with GraphPad 10.0 software. One-way ANOVA, Kruskal-Wallis nonparametric analysis or a paired Student’s t test was used as appropriate. A p-value less than 0.05 was considered statistically significant. Results Clinical and laboratory characteristics between the SMPP and Non-SMPP groups A total of 267 subjects (225 Non-SMPP and 42 SMPP) were enrolled in this study. As shown in Table 1, the duration of illness and hospitalization days in the SMPP group were longer than those in the Non-SMPP group (p<0.0005). The proportion of children with fever in the SMPP group (92.86 % vs. 73.33 %, p=0.0062) was also higher than that in the Non-SMPP group. In laboratory findings, compared with Non-SMPP, children with SMPP had decreased peripheral lymphocyte counts(2.11 vs. 3.45, p=0.0007), higher level of erythrocyte sedimentation rate (ESR)(47 mm/h vs. 37 mm/h, p=0.0161), and especially, had significantly higher proportion of CD4 + CXCR4 + T lymphocytes (30.23 % vs. 13.52 %, p<0.0001). However, between the two groups, there were no significant differences in other laboratory parameters, like CRP, LDH, procalcitonin, and including-mycoplasma pneumoniae IgG/M antibody titer. Tofacitinib improved the lung structure and infiltration of inflammatory cells in MPP mice First, we observed the lung structure and lymphocytes infiltration in MPP mice after the administration of Tofacitinib. As shown in Figure 1, compared with untreated mice, the inflammatory lesion and fibrosis in the lungs of MPP mice were significantly improved and lymphocytes infiltration (8.42±6.43 vs. 26.03±5.5, p<0.005) was reduced after the administration of Tofacitinib. As a routine treatment forMPP, Azithromycin could dramatically reduce the occurrence of inflammation in the lungs of MPP mice (12.92±3.24 vs. 26.03±5.5, p<0.05). As an antagonist of CXCR4, AMD3100 could also improve lung fibrosis and inflammatory cells infiltration in MPP mice (13.14±4.93 vs. 26.03±5.5, p<0.05). The results of inflammatory cells change in BALF are shown in Table 2. Tofacitinib could reduce MP-DNA copy and mortality in MPP mice Next, we analyzed the effect of Tofacitinib on the copy of MP-DNA in BALF and the survival of MPP mice. As shown in Table 2, compared to Wild mice, the copy of MP-DNA in the BALF of MPP mice was significantly increased (1243.94±597.35 vs. 24.43±10.20, p<0.001). Nevertheless, Tofacitinib was able to significantly reduce the copy of MP-DNA in the BALF compared to MPP mice (503.44±98.43 vs. 1243.94±597.35, p<0.01), which is similar to the effect of Azithromycin (309.92±101.29 vs. 1243.94±597.35, p<0.005). More importantly, compared to MPP mice, the mortality of mice treated with Tofacitinib was reduced by 50% (the survival proportion increased to 85% from 70%) (Figure 2). In addition, Azithromycin and AMD3100 could also improve the survival of MPP mice, and AMD3100 combined with Tofacitinib could significantly reduce the mortality rate of MPP mice (Figure 2). Tofacitinib could reduce the CXCR4 expression of CD4 + lymphocytes in MPP mice Compared to MPP mice, the proportion of CXCR4 molecules on the surface of CD4 + lymphocytes in peripheral blood was significantly downregulated after treatment with Tofacitinib (53.56±9.82 vs. 20.29±4.95, p<0.001) (Figure 3A, B). Moreover, this effect of Tofacitinib was better than that of CXCR4 antagonist AMD3100 (20.29±4.95 vs. 30.00±5.25, p<0.005). The combination therapy of Tofacitinib with AMD3100 in MPP mice further decreased the CXCR4 expression comparing with Tofacitinib or AMD3100 alone (Figure 3A,B). The results of fluorescence immunohistochemistry also demonstrated that Tofacitinib can significantly reduce the proportion of CD4 + CXCR4 + lymphocytes within lung tissue (Figure 3C). Besides, as shown in Figure 3D, the concentrations of IL-2, IL-6, IL-8,IFN-γ and IP10 in the BALF of MPP mice were markedly increased. Meaningfully, CXCL12, as the sole ligand of CXCR4, also exhibited a notable elevation in the BALF of MPP mice. Tofacitinib administration not only reduced the concentration of inflammatory cytokines but also showed a particularly significant inhibitory effect on CXCL12 (Figure 3D). Discussion Mycoplasma Pneumoniae Pneumonia (MPP) is a common type of community-acquired pneumonia (CAP) in children, and most of the cases are mild and have a good prognosis. However, in recent years, severe MPP (SMPP) has gradually increased, and is often characterized by severe pulmonary manifestations and extrapulmonary complications, including meningitis, cerebral infarction, myocarditis, hepatitis, and more [9,10] . In our study, SMPP accounted for 15.73% like previous report [5] , exhibited fever more frequently, longer course of disease and hospitalization, and the clinical treatment was more challenging. However, more severe extrapulmonary complications were not observed, which may be related to our prompt treatment and the small number of SMPP patients included. Mycoplasma pneumoniae can infect the entire respiratory tract, even affecting the interstitial lung tissue and alveoli. From the perspective of chest imaging, there is little difference in the location of disease between MPP and SMPP. However, in our study, the incidence of interstitial lung disease in children with Secondary Mycoplasma Pneumoniae Pneumonia (SMPP) was higher than that in the Mycoplasma Pneumoniae Pneumonia (MPP) group. (Data not shown) Therefore, in clinical practice, it is important to evaluate the severity of MPP as soon as possible to prevent adverse outcomes. Some studies have found that serum amyloid A (SAA), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and procalcitonin (PCT) are sensitive markers for diagnosing Mycoplasma pneumoniae infection [11] . Despite some differences, these indicators cannot be used to distinguish SMPP from Non-SMPP due to their low specificity. Therefore, these markers need to be interpreted in conjunction with other cytokines and clinical features. Incidentally, in our study, no significant differences were observed in these parameters except for ESR between the SMPP and Non-SMPP group. Notably, compared with that of Non-SMPP, in peripheral blood of SMPP children, the proportion of CD4 + CXCR4 + T cells was significantly increased, which may be served as a potential predictor of SMPP. Of course, further large-scale studies are needed to confirm this. Instead, the peripheral total lymphocyte counts of SMPP children were decreased obviously, suggesting that there is a notable immune dysregulation in SMPP. To our knowledge, cellular immunity plays a pivotal role in the pathogenesis of MPP. CD4 + T cells are associated with the severity of MPP, while CD8 + T cells inhibit the immune response in the lungs [12] . Among them, CXCR4 on the surface of CD4 + T lymphocytes is involved in regulating the immune response in MPP. With the upregulation of CXCR4 expression, the migration and function of CD4 + T immune cells were enhanced, thereby regulating the intensity and duration of the immune response [13] . In CD4 + T cells, high expression of CXCR4 promotes their recruitment to the site of infection and influences the direction of immune responses by regulating the balance between Th1/Th17 and Treg (regulatory T cell). For instance, an increase in CXCR4 + Th17 cells may exacerbate pulmonary inflammation, while impaired homing of Treg mediated by CXCR4 may lead to insufficient immunosuppressive function, which is associated with the immunopathological damage in severe MPP. Additionally, CXCR4 may indirectly inhibit the anti-infective function of CD8 + T cells by altering the local chemokine environment, thereby weakening the ability to clear pathogens [14,15] . This is of great significance for controlling infection, reducing inflammatory damage, and promoting recovery from the illness. Cytokine and chemokine-mediated inflammation and immune responses also play crucial roles in the pathogenicity of Mycoplasma. In this study, we found the levels of IL-6, IL-8, and IP-10 in BALF of MPP mice were higher than those in Non-MPP mice. Interestingly, we also observed a significant increase of Stromal Cell-Derived Factor-1(SDF-1, also known as CXCL12) in BALF of MPP mice. However, it should be noted that SDF-1 is not a direct pathogenic factor of MPP, but rather a factor involved in its pathophysiological process. CXCL12, as the specific ligand of CXCR4, could induced the migration and function of CD4 + CXCR4 + T cells, thereby regulating the intensity and duration of the immune response. In MPP, the interaction between CXCL12 and CXCR4 may participate in regulating the progression of lung inflammation and immune reactions. MPP is a lung infection caused by Mycoplasma pneumoniae, and its treatment generally relies on targeted antibiotics, such as macrolides (e.g., azithromycin, erythromycin) or fluoroquinolones (e.g., levofloxacin). Tofacitinib, a Janus kinase (JAK) inhibitor, is primarily used to treat autoimmune diseases such as rheumatoid arthritis and is not specifically indicated for the treatment of MPP. It’s worth mentioning, our previous research has shown that, in patients with dermatomyositis with MDA5 + antibody and interstitial lung disease who were treated with Tofacitinib, there was a significant downregulation of CXCR4 on the surface of CD4 + T lymphocytes, and Tofacitinib could inhibit the secretion of profibrotic factors (TGF-β) in lung fibroblasts [16] . Meaningfully, in the present study, in-vivo experiments have also demonstrated that Tofacitinib can suppress the immune and inflammatory response in lungs of MPP mice, improve the survival of MPP mice probably by inhibiting the CXCL12/CXCR4 signaling pathway, potentially exerting a therapeutic effect on MPP. This may be attributed to the fact that Tofacitinib, on the one hand, directly reduces CXCR4 transcription by inhibiting the JAK-STAT-NF-κB axis, and on the other hand, indirectly decreases CXCR4 expression by inhibiting JAK1/JAK3 and thereby blocking STAT3/STAT5 phosphorylation. CXCL12, the primary ligand for CXCR4, is predominantly secreted by pulmonary epithelial cells, fibroblasts, and macrophages. Under pathological conditions such as MMP, macrophages significantly upregulate CXCL12 expression in a manner dependent on JAK-STAT (e.g., IFN-γ/JAK1-STAT1) and NF-κB signaling pathways. Tofacitinib not only reduces the release of pro-inflammatory cytokines (e.g., TNF-α, IL-6) from macrophages, thereby diminishing CXCL12 production, but may also suppress STAT3-mediated CXCL12 secretion by epithelial cells. This dual mechanism attenuates the recruitment of CXCR4 + T cells, ultimately contributing to its therapeutic efficacy. There is an interesting phenomenon in our research findings: the combined administration of AMD3100 and Tofacitinib seems to reduce the proportion of CXCR4 + CD4 + T cells more significantly than either treatment alone. This could be attributed to the fact that, on the one hand, Tofacitinib inhibits the secretion of CXCL12 by macrophages and fibroblasts through the JAK-STAT/NF-κB pathway, thereby reducing the concentration of the ligand CXCR4 in the microenvironment (at the transcriptional level), while AMD3100 blocks the residual function of CXCR4 (at the protein level), resulting in "expression-function" dual inhibition. On the other hand, the combination of Tofacitinib and AMD3100 can enhance CD8 + T cell infiltration, subsequently reducing the accumulation of Th17 cells and achieving more pronounced anti-inflammatory effects compared to monotherapy [17,18,19] . How about the effect and mechanism of Tofacitinib on MPP is still an open question. Azithromycin, as a classic drug for the treatment of MPP, has been shown in our research findings to significantly reduce the expression of CXCR4, which may be the result of multiple factors at play. It is possible that azithromycin, by inhibiting bacterial protein synthesis, reduces pathogen-associated molecular patterns (PAMPs), thereby attenuating the activation of NF-κB and JAK-STAT signaling pathways and subsequently decreasing CXCR4 expression [20] . Additionally, azithromycin can exert effects such as macrophage polarization and Th17 inhibition, further reducing the amount of CXCR4 [21] . Our study has the following limitations. Firstly, it is a single-center study with a relatively small number of patients enrolled in the SMPP group. Additionally, some patients may have had undetected co-infection with other pathogens during hospitalization, or some samples for analysis were obtained after treatment, which might have a certain impact on the experimental results. Furthermore, Tofacitinib has only been validated in vivo animal models and has not undergone further in clinic trial. These factors may lead to some research bias, and future validation in larger, multi-center, prospective studies is needed. Most importantly, it remains unclear whether CXCR4 serves as a passive marker of inflammation, or a key molecule actively involved in immune regulation. Our experimental studies have demonstrated that CXCR4 antagonists (such as AMD3100) can alleviate T cell-mediated lung injury and reduce the release of pro-inflammatory cytokines, suggesting their direct involvement in the activation of signaling pathways (such as PI3K/Akt and MAPK). However, in some cases, the upregulation of CXCR4 may merely reflect the stimulation of inflammatory factors (such as the effects of TNF-α or IL-6), rather than being directly pathogenic. Therefore, future research should incorporate conditional gene knockout models or single cell sequencing technology to further elucidate the specific functions of CXCR4 in different T cell subsets. In conclusion, this single-center study revealed that the peripheral proportion of CD4 + CXCR4 + T cells is elevated significantly in SMPP and has some correlation with clinical symptoms and inflammatory markers, suggesting that it may assist pediatricians to evaluate the severity of MPP. As for whether Tofacitinib can serve as an alternative therapeutic option for SMPP, further studies conducted in multiple centers are required. Declarations Data availability statement The original contributions presented in the study are publicly available. Ethics statement The study was approved by Ethics Committee of Ren Ji Hospital Jiading Branch, Shanghai Jiao Tong University School of Medicine (Ethics number:2024K05). The legal guardian of the participant has signed a written informed consent form. Author Contribution Kaiwen Wang conducted experiments, making figures and tables, and drafting the manuscript. Li Guo performed clinical data and bioinformatics analysis. Yongqi Zhang participated in revising the article and supplementing the data. Haiting Yang collected clinical samples. Jiangfeng Zhao designed the experiments and revised the manuscript. All authors contributed to the article and approved the submitted version. Funding This study did not receive any funding support. Conflict of interest This study has no potential commercial or financial conflicts of interest. References Chao Yan , Guan-Hua Xue , Han-Qing Zhao , Yan-Ling Feng , Jing-Hua Cui , Jing Yuan. Current status of Mycoplasma pneumoniae infection in China. World J Pediatr. 2024;20(1):1-4. doi: 10.1007/s12519-023-00783-x. Gretchen L Parrott, Takeshi Kinjo, Jiro Fujita. A compendium for Mycoplasma pneumoniae. Front Microbiol. 2016 Apr 12:7:513. doi: 10.3389/fmicb.2016.00513. Lee KL, Lee CM, Yang TL, et al. Severe Mycoplasma pneumoniae pneumonia requiring intensive care in children, 2010-2019. J Formos Med Assoc. 2021;120(1):281-291. doi:10.1016/j.jfma.2020.08.018. 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Tables Table 1 Comparison of laboratory and clinical characteristics between SMPP and Non-SMPP groups Non-SMPP (n=225) SMPP (n=42) P value Male (n, %) 125 (55.56) 23 (54.76) 0.9243 Age (years) 6.83±3.16 7.71±2.44 0.0829 Hospitalization days 6.09±1.87 8.26±2.78 <0.0001 Disease duration (days) 14.25±7.14 20.84±7.19 0.0004 Fever (n, %） 165 (73.33) 39 (92.86) 0.0062 Cough (n, %） 209 (92.89) 42 (100) 0.0747 WBC (×10 9 /L) 9.15±9.54 8.06±5.71 0.4837 Neutrophils (×10 9 /L) 4.12±2.95 5.21±5.20 0.0668 Lymphocytes (×10 9 /L) 3.45±2.42 2.11±1.11 0.0007 PLT (×10 9 /L) 317±112 320±103 0.9017 ALT (U/L) 17.73±18.43 16.60±14.39 0.7108 AST (U/L) 30.93±20.11 26.15±9.84 0.1379 LDH (U/L) 275.76±132.63 278.18±86.75 0.8785 CRP (mg/L) 13.49±22.40 13.28±20.09 0.9916 ESR (mm/h) 37.87±24.00 47.26±16.06 0.0161 PCT (ng/ml) 0.23±0.75 0.27±0.72 0.7659 Anti-mycoplasma antibody IgG (<20AU/ml) 804.11±1594.84 581.09±1451.59 0.4176 Anti-mycoplasma antibody IgM (<0.7 S/CO) 16.28±47.93 32.50±50.99 0.0557 CD4 + CXCR4 + % 13.52±16.23 30.23±19.56 <0.0001 Notes: SMPP: severe mycoplasma pneumoniae pneumonia; WBC: white blood cell; ALT: alanine aminotransferase; AST: aspartate aminotransferase; CRP: C reactive protein; LDH: lactic dehydrogenase; ESR: erythrocyte sedimentation rate; PCT: procalcitonin. P < 0.05 indicates statistical significance. Table 2 Effects of Tofacitinib on cell classification of BALF Treatment Cell differentiation (%) MP-DNA Macrophages Neutrophils Lymphocytes （copy/mL） Wild 95.68±3.01 1.98±0.33 3.56±0.21 24.43±10.20 MPP 70.81±4.36** 4.43±2.41* 26.03 ± 5.55 **** 1243.94 ±597.35**** MPP+PBS 72.33±2.35 5.45±1.90 22.94±6.02 1193.54±603.10 MPP+AMD3100 84.97±1.39 2.52±0.69 13.14 ± 4.93 ## 783.02±124.22 ## MPP+Tofacitinib 90.11±4.53 2.33±1.30 8.42 ± 6.43 ### 503.44±98.43 ## MPP+Azithromycin 87.03±1.32 1.43±0.64 12.92±3.24 ## 309.92±101.29 ### MPP+Tofa+AMD3100 94.77±2.98 1.23±0.29 4.89±1.22 #### 102.33±45.54 #### The bronchoalveolar lavage was performed on day 14. Data are presented as mean ± SD in the group of 18 mice. The results were obtained in three separate experiments. * indicates a comparison of the percentage with the Wild group. # indicates a comparison of the percentage with the MPP group. 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-6018592","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":443158576,"identity":"de073584-f04b-4cde-a63a-4e404bc821be","order_by":0,"name":"Kaiwen Wang","email":"","orcid":"","institution":"Shanghai Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Kaiwen","middleName":"","lastName":"Wang","suffix":""},{"id":443158581,"identity":"5fa96e79-0e88-4078-a6de-eeb9a00ba775","order_by":1,"name":"Li Guo","email":"","orcid":"","institution":"Shanghai Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Guo","suffix":""},{"id":443158586,"identity":"0ac34dae-250b-4098-a17e-caf12e80b55b","order_by":2,"name":"Yongqi Zhang","email":"","orcid":"","institution":"Pizhou City Maternity and Child Health Care Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yongqi","middleName":"","lastName":"Zhang","suffix":""},{"id":443158588,"identity":"d86f2f9b-71cc-49f3-ab46-cb1f3e76b797","order_by":3,"name":"Haiting Yang","email":"","orcid":"","institution":"Shanghai University of Medicine \u0026Health Sciences","correspondingAuthor":false,"prefix":"","firstName":"Haiting","middleName":"","lastName":"Yang","suffix":""},{"id":443158594,"identity":"561bd366-b0d0-4d97-af9b-c7c1f85fd93c","order_by":4,"name":"Hui Du","email":"","orcid":"","institution":"Shanghai Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Du","suffix":""},{"id":443158596,"identity":"bda31bc8-8027-4582-ba8e-b5e8bda7c8a1","order_by":5,"name":"Jiangfeng Zhao","email":"data:image/png;base64,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","orcid":"","institution":"Shanghai University of Medicine \u0026Health Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jiangfeng","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2025-02-13 01:23:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6018592/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6018592/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80795528,"identity":"49df7c79-69ac-4da9-8a3e-0ff2e25dd989","added_by":"auto","created_at":"2025-04-17 07:35:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":596480,"visible":true,"origin":"","legend":"\u003cp\u003eHistological examination of Tofacitinib's effects on lung structure in MPP Mice. Mice were administered with PBS, AMD3100 (3 mg/kg/day), Tofacitinib (6 mg/kg/day), and Azithromycin (20 mg/kg/day) subcutaneously via osmotic minipumps. Histological examination was performed using Hematoxylin and Eosin (H\u0026amp;E) staining at an original magnification of 10x. Bar = 200 μm. Tofa: Tofacitinib, Azith: Azithromycin.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6018592/v1/4165215016d5336bfdece5de.png"},{"id":80795525,"identity":"ef272687-27a0-49b3-8072-b1028264b007","added_by":"auto","created_at":"2025-04-17 07:35:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94369,"visible":true,"origin":"","legend":"\u003cp\u003eTofacitinib's effects on survival probability of MPP Mice. Mice were administered with PBS, AMD3100 (3 mg/kg/day), Tofacitinib (6 mg/kg/day), Azithromycin (20 mg/kg/day), and Tofacitinib (6 mg/kg/day) comminated AMD3100 (3 mg/kg/day)subcutaneously via osmotic minipumps. The results were obtained in three separate experiments.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6018592/v1/a5b45889a8e54710ba7e00e7.png"},{"id":80796781,"identity":"97de1256-45b3-49cd-b7df-32451b1fff77","added_by":"auto","created_at":"2025-04-17 07:43:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":391251,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Tofacitinib administration on CXCR4 in MPP mice. The flow cytometry fluorescence peak graph (A) and histogram (B) of CXCR4 expression on the surface of CD4\u003csup\u003e+\u003c/sup\u003e T cells in the peripheral blood of MPP mice under different treatment. (C) Expression of CXCR4 in CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes in the lung tissues of MPP mice by Multiple fluorescence immunohistochemistry. (D) The content of cytokines and chemokines in BALF of MPP mice. Mice were administered with PBS, AMD3100 (3 mg/kg/day), Tofacitinib (6 mg/kg/day), Azithromycin (20 mg/kg/day), and Tofacitinib (6 mg/kg/day) comminated AMD3100 (3 mg/kg/day)subcutaneously via osmotic minipumps. Data are presented as mean ± SD in the group of 18 mice. The results were obtained in three separate experiments.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6018592/v1/3c4189a7236338f7e27a73b0.png"},{"id":80910778,"identity":"34a4ca83-81b3-4166-9b68-10a89d22de85","added_by":"auto","created_at":"2025-04-18 16:01:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1553523,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6018592/v1/97c1a364-9019-4646-b14f-5cb901990c8b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTofacitinib treats Mycoplasma Pneumoniae Pneumonia by inhibiting the CXCR4 molecule on the CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMycoplasma pneumoniae pneumonia (MPP) is a common type of community-acquired pneumonia that mainly occurs in children aged 5 and above in China\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The symptoms of MPP are varied, including fever, cough, sore throat, etc., and are also related to acute exacerbation of asthma. Some with worsening symptoms and imaging findings are defined as severe MPP (SMPP), which may lead to serious intrapulmonary and extrapulmonary complications, such as lung abscesses, bronchiolitis obliterans, bronchiectasis, pulmonary embolism, myocarditis and encephalitis\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWith the lifting of non-pharmaceutical interventions against COVID-19, the infection rate of Mycoplasma pneumoniae (MP) in children and the number of hospitalizations related to MP increased sharply in 2023\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. The clinical manifestations of the affected children were heterogeneous. Although most children were mild and self-limiting, severe, and refractory cases were also on the rise, accounting for up to 15% according to the latest reports\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Early diagnosis, accurate evaluation and effective treatment are of great significance in reducing the incidence and sequelae of SMPP.\u003c/p\u003e \u003cp\u003eThe diagnosis of MPP is not difficult, but there is a lack of potential biomarkers for clinical assessment of the disease condition and phenotype. Currently, commonly used indicators such as blood leukocytes, C-reactive protein (CRP), lactate dehydrogenase (LDH), ferritin, or certain cytokines are not highly specific or sensitive\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. The pathogenesis of MPP mainly involves direct damage caused by MP and abnormal immune response of the host to MP, with the immune activation of T lymphocytes being considered as a crucial factor. Additionally, studies have shown that in the lungs of MPP children with pneumonia as the primary symptom, there is significant infiltration of Mycoplasma-specific Th1 and Th2 cells, which is mainly driven by chemokines and their receptors \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Another research suggested that CXCR4 may play a role in regulating the migration of inflammatory cells in MPP. When MP infects the respiratory tract, CXCL12 (the ligand of CXCR4) is upregulated at the site of infection, attracting CXCR4 positive inflammatory cells to migrate to the infected area\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. These cells participate in immune responses and pathogen clearance by releasing inflammatory mediators and cytokines. However, the expression and specific mechanisms of CXCR4 in MPP are not fully elucidated. This study further explored the expression significance and regulatory mechanisms of CXCR4 in MPP and preliminarily investigated the therapeutic potential of Tofacitinib targeting CXCR4 in MPP mice.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003ePatients\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 267 MPP children who underwent physical examination were prospectively enrolled in the pediatric department of Jiading District Central Hospital from November 2023 to May 2024. The inclusion criteria for MPP children are: (1) Children aged 1-12 years with pneumonia shown on chest imaging and meeting the diagnostic criteria for community-acquired pneumonia (CAP); (2) Positive MP nucleic acid test (collected from nasopharyngeal aspirates) or a fourfold or more increase in serum anti-MP IgM titer during the non-acute phase. The exclusion criteria are: (1) Children with heart disease, congenital immune deficiency, HIV-positive status, or malignant tumors; (2) The co-presence of other pathogens in the child\u0026apos;s body fluids or blood detected by culture, or viral antigen test, or interferon-\u0026gamma; release assays, etc.; (3) Children who have a history of taking immunosuppressants, bronchodilators, or leukotriene receptor antagonists within 2 weeks prior to admission. The study was approved by Ethics Committee of Ren Ji Hospital Jiading Branch, Shanghai Jiao Tong University School of Medicine (Ethics number:2024K05). \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe cultivation of\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eMycoplasma pneumoniae\u003c/p\u003e\n\u003cp\u003eWeigh out 30.8 g of ATCC Medium: 2611 (Shandong Top-Bioengineering Co., Ltd.，China) and add it to 780 ml of deionized water. After heating to dissolve, aliquot the solution. Then, perform autoclaving at 121\u0026deg;C for 15 minutes. Allow it to cool below 50\u0026deg;C. In a sterile environment, add 50 ml of CMRL-1066 tissue culture medium (Amylet Scientific, China) that has been sterilized by filtration, as well as 170 ml of fetal bovine serum (Gibco, USA) that has been inactivated at 55\u0026deg;C for 1 hour. Mix well, aliquot, and set aside for future use. The Mycoplasma pneumoniae strain (ATCC\u003csup\u003e\u0026reg;\u0026nbsp;\u003c/sup\u003e15531\u003csup\u003eTM\u003c/sup\u003e, Type Culture Collection, USA) was diluted with 0.5 mL PPLO broth (Pleuropneumonia-Like Organisms, BD Difco\u0026trade; 211414) and inoculated into liquid culture medium. The culture was incubated at 37\u0026deg;C under 5% CO₂ for 24-72 hours. Subculturing was performed when turbidity was observed in the liquid medium. The bacterial concentration was determined using the Color Changing Units (CCU) assay before subsequent experimental use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnimal Model and Treatment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6 mice weighing 20-25 g (Shanghai Lab. Animal Research Center, China) were anesthetized by intraperitoneal injection of 3 mg phenobarbital sodium (Sangon Biotech (Shanghai) Co., Ltd, China). 0.2 ml of Mycoplasma pneumoniae liquid (ATCC\u003csup\u003e\u0026reg;\u0026nbsp;\u003c/sup\u003e15531\u003csup\u003eTM\u003c/sup\u003e, Type Culture Collection, USA) at logarithmic growth phase with 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e color changing units (ccu)/ml was dripped into the nasal cavity. After 14 days, most of the mice developed respiratory symptoms, and were pathologically confirmed to have pneumonia. An osmotic minipump (Alza Pharmaceuticals, Palo Alto, CA, USA) containing 200 \u0026mu;l saline (respectively with 3 mg/kg/day AMD3100, Sigma-Aldrich, USA; 6 mg/kg/day Tofacitinib, MedChemExpress, China; 20 mg/kg/day Azithromycin, MedChemExpress, China) was implanted subcutaneously into the left suprascapular region through an incision at the base of neck. According to the manufacturer\u0026apos;s guidelines, AMD3100 was administered continuously from day 1 to day 14, Tofacitinib from day 3 to day 14, and Azithromycin from day 3 to day 7. As a control experiment, a minipump containing only saline was also used.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFlow cytometry\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBlood was collected from the peripheral vein of children with MPP or the retro-orbital sinus of mice (on the 14th day after treatment) and then stained with FIFC-CD4, PerCP-CD3 and APC-CXCR4 (BD Biosciences- Pharmingen, San Diego, California, USA). The CXCR4 expression on the surface of CD4\u003csup\u003e+\u003c/sup\u003e lymphocytes was measured using Cellquest software on the FACS Calibur flow cytometer (BD Biosciences-Pharmingen, San Diego, California, USA).\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHE staining\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe lungs of mice (on the 14th day after treatment) were fixed with 0.5 ml of 10% formalin (MACKLIN, China) \u0026nbsp;and then embedded in paraffin. The paraffin blocks were cut into 3-4 \u0026mu;m sections, dewaxed and dried. The slides were stained in hematoxylin (MACKLIN, China) for 8 min, followed by rinsing and immersion in eosin solution (MACKLIN, China) for 4 min. Afterwards, the slides were dehydrated and cleared by immersing in alcohol of different concentrations and xylene sequentially. Finally, the slides were mounted with neutral balsam and observed under a microscope to assess the lung structure. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMultiple fluorescence immunohistochemistry (TSA technology)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter removing the blocking solution from the slice, rabbit anti-mouse CD4 antibody (1:1500, abcam, United Kingdom) and rabbit anti-mouse CXCR4 antibody (1:300, abcam, United Kingdom) were incubated in a humid chamber at 37\u0026deg;C for 2 hours.\u0026nbsp;Subsequently, HRP-Goat Anti-Rabbit IgG H\u0026amp;L (abcam, 1:200) was incubated in the dark at room temperature for 30 min, and then TSA working solution (GEPbio, Shanghai Guipu Biotechnology Co., Ltd., China) was probed for 10 min. The panoramic digital scanning system (KFbio, Ningbo Jiangfeng Biotechnology Information Co., Ltd.，China) were used for analysis after incubating with DAPI staining solution for 10 min.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBronchoalveolar lavage and cell classification\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe distal end of the mouse trachea was ligated, and an intravenous indwelling needle was inserted into the trachea. After connecting the syringe, the trachea was gently lavaged with 1 ml saline for 5 times and followed by collecting the lavage fluid 3 times. After centrifugation at 1200 rpm for 5 min, the precipitate was resuspended in 1 ml liquid for cell counting, and then stained with Diff-Quick (Baxter, Miami, USA) for cell classification.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMeasurement of Mycoplasma DNA in BALF\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMP-DNA was performed using the FQ-PCR kit (Da\u0026apos;an Gene Co., Ltd., China). Specimen processing steps are as follows: i. Four volumes of saline were added to the bronchoalveolar lavage fluid (BALF), which was then repeatedly pipetted with a 1 ml pipette tip and left in \u0026nbsp;4\u0026deg;C overnight to ensure complete liquefaction of the BALF; ii. After mixing, 1 ml of the mixture was aspirated into a 1.5 ml tube and centrifuged at 12,000 rpm for 5 min; iii. Discarded the supernatant and add 50 \u0026mu;l DNA extraction buffer, which was thoroughly mixed and subjected to constant temperature treatment at 100\u0026deg;C for 10 min; iv. The mixture was centrifuged at 12,000 rpm for 5 min and prepared for use.\u0026nbsp;The PCR amplification and data processing \u0026nbsp;were conducted according to the manufacturer\u0026apos;s instructions. using the PEGeneAmp 5700 system (ABI, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnalysis of cytokines and chemokines in BALF\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e24 inflammatory factors in mice were analyzed with the MILLIPLEX\u0026reg; Mouse Cytokine/Chemokine Magnetic Bead kit (Millipore, USA), following the instructions strictly and using Bio-Plex\u0026reg; Manager\u0026trade; Software 6.0 (Bio-Plex, USA) for analysis. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data analysis were performed with GraphPad 10.0 software. One-way ANOVA, Kruskal-Wallis nonparametric analysis or a paired Student\u0026rsquo;s t test was used as appropriate. A p-value less than 0.05 was considered statistically significant.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eClinical and laboratory characteristics between the SMPP and Non-SMPP groups\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 267 subjects (225 Non-SMPP and 42 SMPP) were enrolled in this study. As shown in Table 1, the duration of illness and hospitalization days in the SMPP group were longer than those in the Non-SMPP group (p\u0026lt;0.0005). The proportion of children with fever in the SMPP group (92.86 % vs. 73.33 %, p=0.0062) was also higher than that in the Non-SMPP group. In laboratory findings, compared with Non-SMPP, children with SMPP had decreased peripheral lymphocyte counts(2.11 vs. 3.45, p=0.0007), higher level of erythrocyte sedimentation rate (ESR)(47 mm/h vs. 37 mm/h, p=0.0161), and especially, had significantly higher proportion of CD4\u003csup\u003e+\u003c/sup\u003eCXCR4\u003csup\u003e+\u003c/sup\u003e T lymphocytes (30.23 % vs. 13.52 %, p\u0026lt;0.0001). However, between the two groups, there were no significant differences in other laboratory parameters, like CRP, LDH, procalcitonin, and including-mycoplasma pneumoniae IgG/M antibody titer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTofacitinib improved the lung structure and infiltration of inflammatory cells in MPP mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFirst, we observed the lung structure and lymphocytes infiltration in MPP mice after the administration of Tofacitinib. As shown in Figure 1, compared with untreated mice, the inflammatory lesion and fibrosis in the lungs of MPP mice were significantly improved and lymphocytes infiltration (8.42\u0026plusmn;6.43 vs. 26.03\u0026plusmn;5.5, p\u0026lt;0.005) was reduced after the administration of Tofacitinib. As a routine treatment forMPP, Azithromycin could dramatically reduce the occurrence of inflammation in the lungs of MPP mice (12.92\u0026plusmn;3.24 vs. 26.03\u0026plusmn;5.5, p\u0026lt;0.05). As an antagonist of CXCR4, AMD3100 could also improve lung fibrosis and inflammatory cells infiltration in MPP mice (13.14\u0026plusmn;4.93 vs. 26.03\u0026plusmn;5.5, p\u0026lt;0.05). The results of inflammatory cells change in BALF are shown in Table 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTofacitinib could reduce MP-DNA copy and mortality in MPP mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNext, we analyzed the effect of Tofacitinib on the copy of MP-DNA in BALF and the survival of MPP mice. As shown in Table 2, compared to Wild mice, the copy of MP-DNA in the BALF of MPP mice was significantly increased (1243.94\u0026plusmn;597.35 vs. 24.43\u0026plusmn;10.20, p\u0026lt;0.001). Nevertheless, Tofacitinib was able to significantly\u0026nbsp;reduce the copy of MP-DNA in the BALF compared to MPP mice (503.44\u0026plusmn;98.43 vs. 1243.94\u0026plusmn;597.35, p\u0026lt;0.01), which is similar to the effect of Azithromycin (309.92\u0026plusmn;101.29 vs. 1243.94\u0026plusmn;597.35, p\u0026lt;0.005). More importantly, compared to MPP mice, the mortality of mice treated with Tofacitinib was reduced by 50% (the survival proportion increased to 85% from 70%) (Figure 2). In addition, Azithromycin and AMD3100 could also improve the survival of MPP mice, and AMD3100 combined with Tofacitinib could significantly reduce the mortality rate of MPP mice (Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTofacitinib could reduce the CXCR4 expression of CD4\u003csup\u003e+\u003c/sup\u003e lymphocytes in MPP mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCompared to MPP mice, the proportion of CXCR4 molecules on the surface of CD4\u003csup\u003e+\u003c/sup\u003e lymphocytes in peripheral blood was significantly downregulated after treatment with Tofacitinib (53.56\u0026plusmn;9.82 vs. 20.29\u0026plusmn;4.95, p\u0026lt;0.001) (Figure 3A, B). Moreover, this effect of Tofacitinib was better than that of CXCR4 antagonist AMD3100 \u0026nbsp;(20.29\u0026plusmn;4.95 vs. 30.00\u0026plusmn;5.25, p\u0026lt;0.005). The combination therapy of Tofacitinib with AMD3100 in MPP mice further decreased the CXCR4 expression comparing with Tofacitinib or AMD3100 alone \u0026nbsp;(Figure 3A,B). The results of fluorescence immunohistochemistry also demonstrated that Tofacitinib can significantly reduce the proportion of CD4\u003csup\u003e+\u003c/sup\u003eCXCR4\u003csup\u003e+\u003c/sup\u003e lymphocytes within lung tissue (Figure 3C). Besides, as shown in Figure 3D, the concentrations of IL-2, IL-6, IL-8,IFN-\u0026gamma; and IP10 in the BALF of MPP mice were markedly increased. Meaningfully, CXCL12, as the sole ligand of CXCR4, also exhibited a notable elevation in the BALF of MPP mice. Tofacitinib administration not only reduced the concentration of inflammatory cytokines but also showed a particularly significant inhibitory effect on CXCL12 (Figure 3D).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMycoplasma Pneumoniae Pneumonia (MPP) is a common type of community-acquired pneumonia (CAP) in children, and most of the cases are mild and have a good prognosis. However, in recent years, severe MPP (SMPP) has gradually increased, and is often characterized by severe pulmonary manifestations and extrapulmonary complications, including meningitis, cerebral infarction, myocarditis, hepatitis, and more\u003csup\u003e[9,10]\u003c/sup\u003e. In our study, SMPP accounted for 15.73% like previous report\u003csup\u003e[5]\u003c/sup\u003e, \u0026nbsp;exhibited fever more frequently, longer course of disease and hospitalization, and the clinical treatment was more challenging. However, more severe extrapulmonary complications were not observed, which may be related to our prompt treatment and the small number of SMPP patients included. Mycoplasma pneumoniae can infect the entire respiratory tract, even affecting the interstitial lung tissue and alveoli. From the perspective of chest imaging, there is little difference in the location of disease between MPP and SMPP. However, in our study, the incidence of interstitial lung disease in children with Secondary Mycoplasma Pneumoniae Pneumonia (SMPP) was higher than that in the Mycoplasma Pneumoniae Pneumonia (MPP) group. (Data not shown) Therefore, in clinical practice, it is important to evaluate the severity of MPP as soon as possible to prevent adverse outcomes.\u003c/p\u003e\n\u003cp\u003eSome studies have found that serum amyloid A (SAA), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and procalcitonin (PCT) are sensitive markers for diagnosing Mycoplasma pneumoniae infection\u003csup\u003e[11]\u003c/sup\u003e. Despite some differences, these indicators cannot be used to distinguish SMPP from Non-SMPP due to their low specificity. Therefore, these markers need to be interpreted in conjunction with other cytokines and clinical features. Incidentally, in our study, no significant differences were observed in these parameters except for ESR between the SMPP and Non-SMPP group. Notably, compared with that of Non-SMPP, in peripheral blood of SMPP children, the proportion of CD4\u003csup\u003e+\u003c/sup\u003eCXCR4\u003csup\u003e+\u003c/sup\u003eT cells was significantly increased, which may be served as a potential predictor of SMPP. Of course, further large-scale studies are needed to confirm this. Instead, the peripheral total lymphocyte counts of SMPP children were decreased obviously, suggesting that there is a notable immune dysregulation in SMPP. To our knowledge, cellular immunity plays a pivotal role in the pathogenesis of MPP. CD4\u003csup\u003e+\u003c/sup\u003e T cells are associated with the severity of MPP, while CD8\u003csup\u003e+\u003c/sup\u003e T cells inhibit the immune response in the lungs\u003csup\u003e[12]\u003c/sup\u003e. Among them, CXCR4 on the surface of CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes is involved in regulating the immune response in MPP. With the upregulation of CXCR4 expression, the migration and function of CD4\u003csup\u003e+\u003c/sup\u003e T immune cells were enhanced, thereby regulating the intensity and duration of the immune response\u003csup\u003e[13]\u003c/sup\u003e. In CD4\u003csup\u003e+\u003c/sup\u003e T cells, high expression of CXCR4 promotes their recruitment to the site of infection and influences the direction of immune responses by regulating the balance between Th1/Th17 and Treg (regulatory T cell). For instance, an increase in CXCR4\u003csup\u003e+\u003c/sup\u003e Th17 cells may exacerbate pulmonary inflammation, while impaired homing of Treg mediated by CXCR4 may lead to insufficient immunosuppressive function, which is associated with the immunopathological damage in severe MPP. Additionally, CXCR4 may indirectly inhibit the anti-infective function of CD8\u003csup\u003e+\u003c/sup\u003e T cells by altering the local chemokine environment, thereby weakening the ability to clear pathogens\u003csup\u003e[14,15]\u003c/sup\u003e. This is of great significance for controlling infection, reducing inflammatory damage, and promoting recovery from the illness.\u003c/p\u003e\n\u003cp\u003eCytokine and chemokine-mediated inflammation and immune responses also play crucial roles in the pathogenicity of Mycoplasma. In this study, we found the levels of IL-6, IL-8, and IP-10 in BALF of MPP mice were higher than those in Non-MPP mice. Interestingly, we also observed a significant increase of Stromal Cell-Derived Factor-1(SDF-1, also known as CXCL12) in BALF of MPP mice. However, it should be noted that SDF-1 is not a direct pathogenic factor of MPP, but rather a factor involved in its pathophysiological process. CXCL12, as the specific ligand of CXCR4, could induced the migration and function of CD4\u003csup\u003e+\u003c/sup\u003eCXCR4\u003csup\u003e+\u003c/sup\u003e T cells, thereby regulating the intensity and duration of the immune response. In MPP, the interaction between CXCL12 and CXCR4 may participate in regulating the progression of lung inflammation and immune reactions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMPP is a lung infection caused by Mycoplasma pneumoniae, and its treatment generally relies on targeted antibiotics, such as macrolides (e.g., azithromycin, erythromycin) or fluoroquinolones (e.g., levofloxacin). Tofacitinib, a Janus kinase (JAK) inhibitor, is primarily used to treat autoimmune diseases such as rheumatoid arthritis and is not specifically indicated for the treatment of MPP. It\u0026rsquo;s worth mentioning, our previous research has shown that, in patients with dermatomyositis with MDA5\u003csup\u003e+\u003c/sup\u003e antibody and interstitial lung disease who were treated with Tofacitinib, there was a significant downregulation of CXCR4 on the surface of CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes, and \u0026nbsp;Tofacitinib could inhibit the secretion of profibrotic factors (TGF-\u0026beta;) in lung fibroblasts\u003csup\u003e[16]\u003c/sup\u003e. Meaningfully, in the present study, in-vivo experiments have also demonstrated that Tofacitinib can suppress the immune and inflammatory response in lungs of MPP mice, improve the survival of MPP mice probably by inhibiting the CXCL12/CXCR4 signaling pathway, potentially exerting a therapeutic effect on MPP. This may be attributed to the fact that Tofacitinib, on the one hand, directly reduces CXCR4 transcription by inhibiting the JAK-STAT-NF-\u0026kappa;B axis, and on the other hand, indirectly decreases CXCR4 expression by inhibiting JAK1/JAK3 and thereby blocking STAT3/STAT5 phosphorylation. CXCL12, the primary ligand for CXCR4, is predominantly secreted by pulmonary epithelial cells, fibroblasts, and macrophages. Under pathological conditions such as MMP, macrophages significantly upregulate CXCL12 expression in a manner dependent on JAK-STAT (e.g., IFN-\u0026gamma;/JAK1-STAT1) and NF-\u0026kappa;B signaling pathways. Tofacitinib not only reduces the release of pro-inflammatory cytokines (e.g., TNF-\u0026alpha;, IL-6) from macrophages, thereby diminishing CXCL12 production, but may also suppress STAT3-mediated CXCL12 secretion by epithelial cells. This dual mechanism attenuates the recruitment of CXCR4\u003csup\u003e+\u003c/sup\u003e T cells, ultimately contributing to its therapeutic efficacy. There is an interesting phenomenon in our research findings: the combined administration of AMD3100 and Tofacitinib seems to reduce the proportion of CXCR4\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells more significantly than either treatment alone. This could be attributed to the fact that, on the one hand, Tofacitinib inhibits the secretion of CXCL12 by macrophages and fibroblasts through the JAK-STAT/NF-\u0026kappa;B pathway, thereby reducing the concentration of the ligand CXCR4 in the microenvironment (at the transcriptional level), while AMD3100 blocks the residual function of CXCR4 (at the protein level), resulting in \u0026quot;expression-function\u0026quot; dual inhibition. On the other hand, the combination of Tofacitinib and AMD3100 can enhance CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration, subsequently reducing the accumulation of Th17 cells and achieving more pronounced anti-inflammatory effects compared to monotherapy\u003csup\u003e[17,18,19]\u003c/sup\u003e. How about the effect and mechanism of Tofacitinib on MPP is still an open question. Azithromycin, as a classic drug for the treatment of MPP, has been shown in our research findings to significantly reduce the expression of CXCR4, which may be the result of multiple factors at play. It is possible that azithromycin, by inhibiting bacterial protein synthesis, reduces pathogen-associated molecular patterns (PAMPs), thereby attenuating the activation of NF-\u0026kappa;B and JAK-STAT signaling pathways and subsequently decreasing CXCR4 expression\u003csup\u003e[20]\u003c/sup\u003e. Additionally, azithromycin can exert effects such as macrophage polarization and Th17 inhibition, further reducing the amount of CXCR4\u003csup\u003e[21]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur study has the following limitations. Firstly, it is a single-center study with a relatively small number of patients enrolled in the SMPP group. Additionally, some patients \u0026nbsp; may have had undetected co-infection with other pathogens during hospitalization, or some samples for analysis were \u0026nbsp;obtained after treatment, which might have a certain impact on the experimental results. Furthermore, Tofacitinib has only been validated in vivo animal models and has not undergone further in clinic trial. These factors may lead to some research bias, and future validation in larger, multi-center, prospective studies is needed. Most importantly, it remains unclear whether CXCR4 serves as a passive marker of inflammation, or a key molecule actively involved in immune regulation. Our experimental studies have demonstrated that CXCR4 antagonists (such as AMD3100) can alleviate T cell-mediated lung injury and reduce the release of pro-inflammatory cytokines, suggesting their direct involvement in the activation of signaling pathways (such as PI3K/Akt and MAPK). However, in some cases, the upregulation of CXCR4 may merely reflect the stimulation of inflammatory factors (such as the effects of TNF-\u0026alpha; or IL-6), rather than being directly pathogenic. Therefore, future research should incorporate conditional gene knockout models or single cell sequencing technology to further elucidate the specific functions of CXCR4 in different T cell subsets.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this single-center study revealed that the peripheral proportion of CD4\u003csup\u003e+\u003c/sup\u003eCXCR4\u003csup\u003e+\u003c/sup\u003e T cells is elevated significantly in SMPP and has some correlation with clinical symptoms and inflammatory markers, suggesting that it may assist pediatricians to evaluate the severity of MPP. As for whether Tofacitinib can serve as an alternative therapeutic option for SMPP, further studies conducted in multiple centers are required.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are publicly available.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by Ethics Committee of Ren Ji Hospital Jiading Branch, Shanghai Jiao Tong University School of Medicine (Ethics number:2024K05). The legal guardian of the participant has signed a written informed consent form.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKaiwen Wang conducted experiments, making figures and tables, and drafting the manuscript. Li Guo performed clinical data and bioinformatics analysis. Yongqi Zhang participated in revising the article and supplementing the data. Haiting Yang collected clinical samples. Jiangfeng Zhao designed the experiments and revised the manuscript. All authors contributed to the article and approved the submitted version.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not receive any funding support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has no potential commercial or financial conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChao Yan , Guan-Hua Xue , Han-Qing Zhao , Yan-Ling Feng , Jing-Hua Cui , Jing Yuan. Current status of Mycoplasma pneumoniae infection in China. World J Pediatr. 2024;20(1):1-4. doi: 10.1007/s12519-023-00783-x. \u003c/li\u003e\n\u003cli\u003eGretchen L Parrott, Takeshi Kinjo, Jiro Fujita. A compendium for Mycoplasma pneumoniae. Front Microbiol. 2016 Apr 12:7:513. doi: 10.3389/fmicb.2016.00513.\u003c/li\u003e\n\u003cli\u003eLee KL, Lee CM, Yang TL, et al. Severe Mycoplasma pneumoniae pneumonia requiring intensive care in children, 2010-2019. J Formos Med Assoc. 2021;120(1):281-291. doi:10.1016/j.jfma.2020.08.018.\u003c/li\u003e\n\u003cli\u003ePatrick M Meyer Sauteur , Michael L Beeton. ancet Microbe. Mycoplasma pneumoniae: delayed re-emergence after COVID-19 pandemic restrictions. Lancet Microbe. 2024;5(2):e100-e101. doi: 10.1016/S2666-5247(23)00344-0\u003c/li\u003e\n\u003cli\u003eWang X, Li M, Luo M, Luo Q, Kang L, Xie H, et al. Mycoplasma pneumoniae triggers pneumonia epidemic in autumn and winter in Beijing: a multicentre, population-based epidemiological study between 2015 and 2020. Emerg Microbes Infect. 2022; 11:1508\u0026ndash;17. doi: 10.1080/22221751.2022.2078228. \u003c/li\u003e\n\u003cli\u003eFan F, Lv J, Yang Q, Jiang F. Clinical characteristics and serum inflammatory markers of community-acquired mycoplasma pneumonia in children. Clin Respir J. 2023 Jul;17(7):607-617. doi: 10.1111/crj.13620. \u003c/li\u003e\n\u003cli\u003eYifan Zhu , Yingying Luo , Ling Li , Xinyi Jiang , Yi Du , Jing Wang , et al. Immune response plays a role in Mycoplasma pneumoniae pneumonia. Front Immunol. 2023;14:1189647. doi: 10.3389/fimmu.2023.1189647.\u003c/li\u003e\n\u003cli\u003eYi-Chen Lee, Chih-Hao Chang, Wei-Ju Lee, Ta-Yu Liu, Chih-Min Tsai, Ti-An Tsai, et al. Altered chemokine profile in Refractory Mycoplasma pneumoniae pneumonia infected children. J Microbiol Immunol Infect. 2021;54(4):673-679.doi: 10.1016/j.jmii.2020.03.030.\u003c/li\u003e\n\u003cli\u003eJin X, Zou Y, Zhai J, Liu J, Huang B. Refractory Mycoplasma pneumoniae pneumonia with concomitant acute cerebral infarction in a child: a case report and literature review. Medicine (Baltimore). 2018;97(13):e0103. doi: 10.1097/MD.0000000000010103.\u003c/li\u003e\n\u003cli\u003eSaraya T. Mycoplasma pneumoniae infection: basics. J Gen Fam Med. 2017;18(3):118‐125. doi: 10.1002/jgf2.15.\u003c/li\u003e\n\u003cli\u003eJiang Y, Wang W, Zhang Z, et al. Serum amyloid a, C‐reactive protein, and procalcitonin levels in children with Mycoplasma pneumoniae infection. J Clin Lab Anal. 2022;36(3):e24265. doi: 10.1002/jcla.24265.\u003c/li\u003e\n\u003cli\u003eJones HP, Tabor L, Sun X, Woolard MD, Simecka JW. Depletion of CD8+ T cells exacerbates CD4+ Th cell‐associated inflammatory lesions during murine mycoplasma respiratory disease. J Immunol. 2002;168(7):3493‐3501. doi:10.4049/jimmunol.168.7.3493.\u003c/li\u003e\n\u003cli\u003eT Nanki, P E Lipsky. Cutting edge: stromal cell-derived factor-1 is a costimulator for CD4+ T cell activation. J Immunol. 2000;164(10):5010-4. doi: 10.4049/jimmunol.164.10.5010.\u003c/li\u003e\n\u003cli\u003eXiulian Zhang, Xueliang Li, Wei Ma, Fangying Liu, Pinxian Huang, Lei Wei, et al. Astragaloside IV restores Th17/Treg balance via inhibiting CXCR4 to improve chronic obstructive pulmonary disease. Immunopharmacol Immunotoxicol. 2023;45(6):682-691.doi: 10.1080/08923973.2023.2228479.\u003c/li\u003e\n\u003cli\u003eZhang L, Liu K, Duan X, Zhou S, Jia H, You Y, et al. CXCL12/CXCR4 axis mediates CD8 \u003csup\u003e+\u003c/sup\u003e T cell overactivation in the progression of viral myocarditis. 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J Investig Dermatol Symp Proc. 2013;16(1):S70-2. doi: 10.1038/jidsymp.2013.29.\u003c/li\u003e\n\u003cli\u003eYang-Shan Fu, Xue-Qiong Duan, Ke-Run Cheng, Yan-Yan-Fei, Lin Liu, Hong-Dan Duan, et al. Geraniol relieves mycoplasma pneumonia infection-induced lung injury in mice through the regulation of ERK/JNK and NF-\u0026kappa;B signaling pathways. J Biochem Mol Toxicol. 2022;36(4):e22984. doi: 10.1002/jbt.22984.\u003c/li\u003e\n\u003cli\u003eXin Yang, Lihua Gao. Effect of azithromycin combined with fluticasone propionate aerosol inhalation on immune function in children with chronic cough caused by Mycoplasma pneumoniae infection. Eur J Pediatr. 2025 Jan 27;184(2):155. doi: 10.1007/s00431-024-05961-1.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 Comparison of laboratory and clinical characteristics between SMPP and Non-SMPP groups\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eNon-SMPP (n=225)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eSMPP (n=42)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eMale (n, %)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e125 (55.56)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e23 (54.76)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.9243\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e6.83\u0026plusmn;3.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e7.71\u0026plusmn;2.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.0829\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eHospitalization days\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e6.09\u0026plusmn;1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e8.26\u0026plusmn;2.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0.0001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eDisease duration\u0026nbsp;(days)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e14.25\u0026plusmn;7.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e20.84\u0026plusmn;7.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e0.0004\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eFever (n,\u0026nbsp;%）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e165 (73.33)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e39 (92.86)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e0.0062\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eCough (n,\u0026nbsp;%）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e209 (92.89)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e42 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.0747\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eWBC (\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e9.15\u0026plusmn;9.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e8.06\u0026plusmn;5.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.4837\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eNeutrophils (\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e4.12\u0026plusmn;2.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e5.21\u0026plusmn;5.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.0668\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eLymphocytes (\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e3.45\u0026plusmn;2.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2.11\u0026plusmn;1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e0.0007\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003ePLT (\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e317\u0026plusmn;112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e320\u0026plusmn;103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.9017\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eALT (U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e17.73\u0026plusmn;18.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e16.60\u0026plusmn;14.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.7108\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eAST (U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e30.93\u0026plusmn;20.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e26.15\u0026plusmn;9.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.1379\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eLDH (U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e275.76\u0026plusmn;132.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e278.18\u0026plusmn;86.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.8785\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eCRP (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e13.49\u0026plusmn;22.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e13.28\u0026plusmn;20.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.9916\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eESR (mm/h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e37.87\u0026plusmn;24.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e47.26\u0026plusmn;16.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e0.0161\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003ePCT (ng/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e0.23\u0026plusmn;0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.27\u0026plusmn;0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.7659\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eAnti-mycoplasma antibody IgG (\u0026lt;20AU/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e804.11\u0026plusmn;1594.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e581.09\u0026plusmn;1451.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.4176\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eAnti-mycoplasma antibody IgM (\u0026lt;0.7 S/CO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e16.28\u0026plusmn;47.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e32.50\u0026plusmn;50.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e0.0557\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 236px;\"\u003e\n \u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003eCXCR4\u003csup\u003e+\u003c/sup\u003e%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e13.52\u0026plusmn;16.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e30.23\u0026plusmn;19.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0.0001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eNotes: SMPP: severe mycoplasma pneumoniae pneumonia; WBC: white blood cell; ALT: alanine aminotransferase; AST: aspartate aminotransferase; CRP: C reactive protein; LDH: lactic dehydrogenase; ESR: erythrocyte sedimentation rate; PCT: procalcitonin. P \u0026lt; 0.05 indicates statistical significance.\u003c/p\u003e\n\u003cp\u003eTable 2\u0026nbsp;Effects of\u0026nbsp;Tofacitinib\u0026nbsp;on cell classification of BALF\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 150px;\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 327px;\"\u003e\n \u003cp\u003eCell differentiation (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003eMP-DNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003eMacrophages\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003eNeutrophils\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003eLymphocytes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e（copy/mL）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e95.68\u0026plusmn;3.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e1.98\u0026plusmn;0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e3.56\u0026plusmn;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e24.43\u0026plusmn;10.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eMPP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e70.81\u0026plusmn;4.36**\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.43\u0026plusmn;2.41*\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e26.03\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026plusmn;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e5.55\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e****\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e1243.94\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026plusmn;597.35****\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eMPP+PBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e72.33\u0026plusmn;2.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e5.45\u0026plusmn;1.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e22.94\u0026plusmn;6.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e1193.54\u0026plusmn;603.10\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eMPP+AMD3100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e84.97\u0026plusmn;1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e2.52\u0026plusmn;0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e13.14\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026plusmn;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e4.93\u003csup\u003e##\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e783.02\u0026plusmn;124.22\u003csup\u003e##\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eMPP+Tofacitinib\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e90.11\u0026plusmn;4.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e2.33\u0026plusmn;1.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e8.42\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026plusmn;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e6.43\u003csup\u003e###\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e503.44\u0026plusmn;98.43\u003csup\u003e##\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eMPP+Azithromycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e87.03\u0026plusmn;1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e1.43\u0026plusmn;0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e12.92\u0026plusmn;3.24\u003csup\u003e##\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e309.92\u0026plusmn;101.29\u003csup\u003e###\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eMPP+Tofa+AMD3100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e94.77\u0026plusmn;2.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e1.23\u0026plusmn;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e4.89\u0026plusmn;1.22\u003csup\u003e####\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e102.33\u0026plusmn;45.54\u003csup\u003e####\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe bronchoalveolar lavage was performed on day 14. Data are presented as mean \u0026plusmn; SD in the group of 18 mice. The results were obtained in three separate experiments. * indicates a comparison of the percentage with the Wild group. # indicates a comparison of the percentage with the MPP group.\u003c/p\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":"Mycoplasma pneumoniae pneumonia, CD4+CXCR4+ lymphocyte, Cytokine, Tofacitinib, Therapy","lastPublishedDoi":"10.21203/rs.3.rs-6018592/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6018592/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eTo compare the differences in CXCR4 expression on the surface of CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes between children with severe Mycoplasma pneumoniae pneumonia (SMPP) and those with common Mycoplasma pneumoniae pneumonia (Non-SMPP) and verify in vitro the potential application value of Tofacitinib as an adjuvant therapy for MPP by affecting the CXCR4 signaling pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e This study recruited 267 children with Mycoplasma pneumoniae pneumonia (MPP), including 42 cases of SMPP and 225 cases of Non-SMPP at Jiading District Central Hospital in Shanghai from 2023 to 2024. Laboratory tests conducted within 24 hours after admission were used as baseline data, and the differences in CD4\u003csup\u003e+\u003c/sup\u003eCXCR4\u003csup\u003e+\u003c/sup\u003e expression between SMPP and Non-SMPP patients were compared. Animal experiments with different medicines were performed to evaluate the mechanism and therapeutic value of Tofacitinib as an adjuvant therapy for MPP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Compared with Non-SMPP, children with SMPP had more fever (p=0.0062), longer durations of hospital stays (p\u0026lt;0.0001), higher level of erythrocyte sedimentation rate (p=0.0161) and especial the proportion of CD4\u003csup\u003e+\u003c/sup\u003eCXCR4\u003csup\u003e+\u003c/sup\u003e T lymphocytes (p\u0026lt;0.0001).However, there were no significant differences in C-reactive protein, procalcitonin and lactate dehydrogenase levels between SMPP and Non-SMPP group. In animal experiments, compared to wild-type mice, the levels of inflammatory cytokines (IL-6, IL-8, IP-10, and IL-2) were significantly elevated in the bronchoalveolar lavage fluid (BALF) of MPP mice. Notably, the sole ligand of CXCR4, CXCL12 was also markedly increased in the BALF of MPP mice. Tofacitinib could significantly reduce the expression of CXCR4 on the surface of CD4\u003csup\u003e+\u003c/sup\u003e cells in MPP mice, as well as decrease the expression of a series of inflammatory cytokines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e In summary, the proportion of CD4\u003csup\u003e+\u003c/sup\u003eCXCR4\u003csup\u003e+\u003c/sup\u003e T cells may be served as a predictive indicator of clinical severity for the MPP. Tofacitinib can reduce the release of inflammatory cytokines by inhibiting the CXCL12/CXCR4 signaling pathway and may be used as an adjuvant therapy for MPP.\u003c/p\u003e","manuscriptTitle":"Tofacitinib treats Mycoplasma Pneumoniae Pneumonia by inhibiting the CXCR4 molecule on the CD4+ T lymphocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 07:35:40","doi":"10.21203/rs.3.rs-6018592/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":"65a0d298-3fd8-45d9-977c-0da2999492c7","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-18T15:53:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-17 07:35:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6018592","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6018592","identity":"rs-6018592","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}