Dual-phase study of CD4+CXCR4+ T cells in Mycoplasma pneumoniae pneumonia: clinical correlations in children and therapeutic exploration with tofacitinib in mice

This study had two primary objectives. First, we aimed to investigate whether differential expression of the chemokine receptor CXCR4 on CD4 + T lymphocytes could serve as a distinguishing immunological feature between pediatric patients with severe Mycoplasma pneumoniae pneumonia (SMPP) and those with on- Non-severe MPP. Second, we sought to explore the therapeutic potential of the JAK inhibitor Tofacitinib in MPP by examining its effects on CXCR4 pathway modulation, using both clinical observations and experimental validation through an established animal model of MPP. We conducted a prospective cohort study involving 267 pediatric patients diagnosed with MPP at Jiading District Central Hospital between 2023 and 2024, comprising 42 SMPP cases and 225 Non-severe MPP cases. Baseline clinical and laboratory parameters were systematically collected within 24 h of hospital admission. Flow cytometry was employed to quantify the percentage of CD4 + T cells expressing CXCR4 (CD4 + CXCR4 + ) in peripheral blood samples by flow cytometry. For mechanistic investigation, we established a murine model of MPP to evaluate the immunomodulatory effects of Tofacitinib. Treatment groups received either vehicle control or Tofacitinib, after which we analyzed bronchoalveolar lavage fluid (BALF) for inflammatory cytokines (IL-6, IL-8, IP-10, IL-2) and CXCL12 levels via ELISA. Additionally, in vitro experiments were performed using the murine lung epithelial cell line MLE-12 to assess the combined effects of Tofacitinib and a CXCR4 inhibitor on key inflammatory signaling pathways (JAK-STAT and NF-κB) through Western blot analysis. Clinical analysis revealed that children with SMPP had significantly prolonged fever duration ( P = 0.0062), extended hospitalization ( P < 0.0001), elevated erythrocyte sedimentation rate ( P = 0.0161), and higher proportions of CD4 + CXCR4 + T lymphocytes ( P < 0.0001) compared to Non-severe MPP patients. However, no statistically significant differences were observed in serum levels of C-reactive protein, procalcitonin, or lactate dehydrogenase between the two groups. In the MPP mouse model, BALF analysis demonstrated marked increases in pro-inflammatory cytokines (IL-6, IL-8, IP-10, IL-2) and CXCL12 ( P < 0.05), all of which were significantly attenuated by Tofacitinib treatment. Furthermore, Tofacitinib administration reduced CXCR4 expression on CD4 + T cells in lung tissues. In vitro experiments confirmed that the combination of Tofacitinib and a CXCR4 inhibitor synergistically suppressed activation of the JAK-STAT and NF-κB pathways. Our findings indicate that elevated CD4 + CXCR4 + T cell proportions may serve as a predictive biomarker for disease severity in pediatric MPP. The experimental data demonstrate that Tofacitinib exerts protective effects in MPP by modulating the CXCL12/CXCR4 axis, leading to reduced inflammatory cytokine production. The observed suppression of JAK-STAT and NF-κB signaling pathways provides mechanistic support for the potential therapeutic application of Tofacitinib, either alone or in combination with CXCR4 inhibition, in the management of MPP. Further clinical studies are warranted to validate these findings and assess the translational potential of this treatment strategy.