3.1 Patients Characteristics
This study retrospectively analyzed the data of 161 patients with pneumonia including 100 cases in the severe pneumonia group and 61 cases in the mild pneumonia group (Table 1). There were no significant differences in age, gender and weight between the two groups. The days of fever and hospitalization in the severe group were significantly longer than those in the mild group.
The red blood cell count (RBC) and hemoglobin level of the severe group decreased significantly, and the white blood cell count (WBC), platelet count and neutrophil percentage were not significantly different between the two groups. There was no significant difference in CRP levels between the two groups, while PCT levels of the severe group were significantly increased.
And in the severe group, the levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) increased significantly, while the levels of albumin were decreased significantly. The levels of fibrinogen and D-dimer in the severe group were also increased significantly.
Among children with severe pneumonia, 51 cases progressed to respiratory failure, 1 case developed acute respiratory distress syndrome (ARDS), 24 cases had pleural effusion, and 28 cases had pulmonary necrosis. 34 cases were treated with non-invasive continuous positive airway pressure and 1 case was treated with invasive mechanical ventilation (endotracheal intubation).
The microorganisms of children with pneumonia were shown in Table 2. Among the 161 children with pneumonia, the detection rate of MP was the highest; the top three detection rates were Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus; the virus detected rate adenovirus ranked first, followed by respiratory syncytial virus. The pathogen detection rate of severe pneumonia group was higher than that of mild pneumonia group (100% vs. 85.24%). The pathogen detection of bacteria, virus, MP and CP showed no statistical difference between mild and severe pneumonia groups (P>0.05, Table 2). In the mild pneumonia group, at most two kinds of pathogenic microorganisms were detected simultaneously in mixed infection patients. In the severe pneumonia group, at most three kinds of pathogenic microorganisms were detected simultaneously in mixed infected patients.
3.2 Both pro-inflammatory and anti-inflammatory cytokines are elevated in BALF of children with severe pneumonia
We used flow cytometry to detect the cytokines levels in BALF. The results showed that the levels of IL-6 (190.89pg/mL, 70.23~512.57pg/mL), IL-10 (5.09pg/ml, 1.25~11.57pg/mL), IL-17A (4.80pg/mL, 1.25~22.16pg/mL) and TNF-α (8.28pg/mL, 1.25~26.27pg/mL) in BALF of children with severe pneumonia were significantly higher than those of children with mild pneumonia (Fig.1a). The levels of IL-2, IL-4 and IFN-γ in BALF were not significantly different between the two groups (P>0.05, Fig.1a).
3.3 The ratios of pro-inflammatory to anti-inflammatory cytokine in BALF of severe group are higher than that of mild group
It is important to maintain a balance between pro-inflammatory and anti-inflammatory cytokines, so we calculated the ratio of proinflammatory cytokines to anti-inflammatory cytokines in BALF of children with pneumonia. In our study, the IL-6/IL-10 ratio (34.48, 13.82~66.67) and TNF-α/IL-10 ratio (0.91, 1.16~2.64) in BALF of severe group were significantly higher than those of mild group (Fig.1b). There was no significant difference in the IL-17A/IL-10 ratio between the two groups (P>0.05, Fig.1b).
3.4 There are more inflammatory cells in BALF of severe group than that of mild group
Next, we analyzed the levels of inflammatory cells in BALF of children with pneumonia. The inflammatory cell count (2525.00*106/L, 1187.50~5100.00*106/L), neutrophil count (1157.70*106/L, 440.90~2726.54*106/L), macrophage count (488.00*106/L, 243.53~849.90*106/L) and lymphocyte count (128.20*106/L, 128.20~861.90*106/L) (Fig.1c) in BALF of children with severe group were significantly higher than those of children with mild group. And, the percentage of neutrophils (51.00%, 29.00~65.00%) (Fig.1d) in BALF of children with severe pneumonia also increased significantly. Interestingly, the percentage of macrophages (21.00%, 11.00~34.00%) (Fig.1d) in BALF of children with severe pneumonia was significantly reduced. No significant difference was found in the percentage of lymphocytes in BALF between the two groups (P>0.05, Fig.1d).
3.5 Analysis of Receiver operator characteristic curves (ROCs)
The ROCs of the cytokines and inflammatory cells in BALF were performed (Table 3). In our study, ROC analysis showed that if IL-6 > 60.37pg/mL, IL-10 > 2.54pg/mL, TNF-α > 13.07pg/mL, IL-6/IL-10 ratio > 29.03, TNF-α/IL-10 ratio > 1.57, total number of inflammatory cells > 3140.00*106/L, neutrophil count > 768.00*106/L, the percentage of neutrophils > 43.00%, the percentage of macrophages ≤ 50.00%, which indicated that the child may be still in critical condition. The other cytokines and inflammatory cells in BALF in our study had no significant identification ability.
3.6 Analysis of Spearman Correlation
We used Spearman correlation analysis to further understand the relationship between cytokines and inflammatory cells in BALF of children with pneumonia (Fig.2). The levels of cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, IFN-γ) in BALF of children with pneumonia were significantly positively correlated in pairs. As shown in Fig.2, IL-6 levels were significantly positively correlated with the levels of IL-10, IL-17A, TNF-α, IFN-γ (r = 0.791, r=0.466, r = 0.810, r = 0.650, respectively). Similarly, IL-10 levels were also significantly correlated with the levels of IL-17A, TNF-α and IFN-γ (r=0.450, r = 0.765, r = 0.701, respectively).
The total number of inflammatory cells in BALF was significantly positively correlated with the levels of IL-6, IL-10, TNF-α and IFN-γ (r=0.576, r=0.507, r=0.481, r=0.341, respectively). There was no statistically significant correlation between the total number of inflammatory cells and the levels of IL-2, IL-4 and IL-17A (P>0.05). The percentage of neutrophils in BALF was significantly positively correlated with the levels of IL-6, IL-10, IL-17A, TNF-α and IFN-γ (r=0.517, r=0.417, r=0.281, r=0.383, r=0.287, respectively). There was no statistically significant correlation between the percentage of neutrophils and the levels of IL-2 and IL-4 (P>0.05). On the contrary, the percentage of macrophages was significantly negatively correlated with the levels of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α and IFN-γ (r= -0.169, r= -0.157, r= -0.525, r= -0.451, r= -0.396, r= -0.462, r= -0.345, respectively). The correlations between the levels of cytokines and the percentage of lymphocytes were not significant (P>0.05). The total number of inflammatory cells in BALF was directly proportional to the percentage of neutrophils (r=0.603), and inversely proportional to the percentage of lymphocytes (r= -0.182) and macrophages (r= -0.441). In addition, the percentage of neutrophils and macrophages were negatively correlated (r= -0.750).