High expression of HMGB1 in children with refractory Mycoplasma pneumoniae pneumonia

Abstract

Background: Increasing numbers of refractory or severe, even fatal, cases of Mycoplasma pneumoniae infections have been reported in recent years. Excessive inflammatory responses play a vital role in the pathogenesis of refractory M. pneumoniae pneumonia (RMPP). HMGB1 is an actively secreted cytokine produced by macrophages and other inflammatory cells that participates in various infectious diseases. The present study aimed to explore the role and clinical significance of HMGB1 in children with RMPP and the potential mechanism of HMGB1 expression.

Methods: Four hundred and fifty-two children diagnosed with M. pneumoniae pneumonia, including 108 children with RMPP, were enrolled from January 2013 to December 2015 at the Children's Hospital of Soochow University. HMGB1, TNF-α, and IL-6 in peripheral blood from RMPP and non-RMPP (NRMPP) cases were detected by real-time PCR and ELISA. Lipid-associated membrane proteins (LAMPs) were extracted from live M. pneumoniae and prepared at different concentrations for stimulation of THP-1 cells. After coculture with LAMPs, HMGB1, TNF-α, IL-6, RAGE, TLR2, and TLR4 in THP-1 cells were detected by real-time PCR.

Results: Occurrences of cough, fever, and abnormal lung signs were more frequent in RMPP cases compared with NRMPP cases (all p < 0.05). Children with RMPP had longer hospital stays than children with NRMPP (p < 0.05). Different distributions of lymphocytes were noted between RMPP and NRMPP cases. HMGB1, TNF-α, and IL-6 levels were significantly higher in RMPP cases compared with NRMPP cases (all p < 0.05). HMGB1 had good diagnostic ability to differentiate RMPP with AUC of 0.876, sensitivity of 0.833, and specificity of 0.824 compared with TNF-α and IL-6. HMGB1 expression in THP-1 cells was increased by stimulation with 10 μg/ml LAMPs. TLR2 expression was increased after stimulation with 6 μg/ml LAMPs. HMGB1 level was positively associated with TNF-α, IL-6, and TLR2 levels.

Conclusions: HMGB1 is a good diagnostic biomarker for differentiating RMPP and NRMPP. LAMPs from M. pneumoniae may induce HMGB1 expression in immune cells through the TLR2 pathway. Further in vitro and in vivo studies are needed for the development of a new treatment strategy to inhibit the HMGB1 pathway, thereby preventing the inflammation in RMPP.

Keywords: HMGB1; Lipid-associated membrane proteins (LAMPs); M. Pneumoniae pneumonia; RMPP.

Fig. 1

HMGB1, TNF-α, IL-6 levels in children with RMPP or NRMPP. (a) Comparison of HMGB1 between RMPP and NRMPP; (b) Comparison of TNF-α between RMPP and NRMPP; (c) Comparison of IL-6 between RMPP and NRMPP. * RMPP: refractory Mycoplasma pneumoniae pneumonia; NRMPP: non-refractory Mycoplasma pneumoniae pneumonia. * p < 0.05; *** p < 0.001

Fig. 2

Associations between inflammatory factors including HMGB1, TNF-α, IL-6 and duration of fever and hospital stay. p < 0.05 was considered statistically significant

Fig. 3

Diagnosis value of HMGB1, TNF-α, IL-6 in children with RMPP. HMGB1 have good diagnostic ability to differentiate RMPP with the AUC of 0.876, the sensitivity of 0.833, and the specificity of 0.824 compared to TNF-α and IL-6

Fig. 4

THP-1 cells stimulated by LAMPs extracted from M. pneumoniae. THP-1 cells were cultured in RPMI 1640 containing 10% FCS, 2 mM l-glutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin. The cell concentration of THP-1 was adjusted to 1 × 10⁶ ml in 24-well plates. Different doses of LAMPs (0 μg/ml, 2 μg/ml, 4 μg/ml, 6 μg/ml, 8 μg/ml and 10 μg/ml) were co-cultured with THP-1 cells for 16 h. (a) Expression of HMGB1, TNF-α, IL-6 induced by LAMPs; (b) Expression of RAGE, TLR2, TLR4 induced by LAMPs. Data are shown as mean ± SEM of 3 independent experiments. * p < 0.05; ** p < 0.01