At present, pneumonia is still one of the reasons that threaten the health of children in the world. The clinical manifestations of children with pneumonia are very complicated due to the infection by a variety of pathogenic microorganisms. As we all known, inflammation is not only related to the invasion of pathogenic microorganisms, but also related to the body’s inflammatory immune response24. We should look for a detection or treatment method for the inflammatory immune response of pneumonia25, so that we can quickly identify the critically ill children and take more effective treatments. The intense inflammatory storm in patient with severe pneumonia has aroused widespread concern. And our study further demonstrates that the cytokines and inflammatory cells in BALF are effective tools for assessing the severity of pneumonia.
Our study shown that compared with children with mild pneumonia, children with severe pneumonia had longer days of fever and hospitalization. The significant decrease of RBC and hemoglobin level in children with severe pneumonia also indicated a decrease of oxygen carrying capacity, which was related to more severe hypoxia symptoms. The inflammatory markers (PCT, LDH) in children with severe pneumonia also increased significantly. And the abnormal liver function and blood coagulation function in children with severe pneumonia suggested that severe patients had damage external lung organ.
Children with severe pneumonia had abnormal inflammatory immune responses. In our study, the levels of IL-6, IL-10, IL-17A, TNF-α and the number of inflammatory cells (including neutrophils, macrophages and lymphocytes) in BALF of children with severe pneumonia were significantly higher than those of children with mild pneumonia. These results were similar to previous study8,26. Interestingly, compared with children with mild pneumonia, the percentage of macrophages in BALF of children with severe pneumonia was decreased relatively, while the percentage of neutrophils increased significantly. It suggests that undue infiltration of neutrophils is an important mechanism of lung inflammation in children with severe pneumonia.
The levels of IL-6 are associated with the severity of infection and inflammation27–30,as well as the severity of adult pneumonia31. In this study, compared with children with mild pneumonia, children with severe pneumonia had more severe inflammatory cell infiltration in lungs. Immune cells, such as activated alveolar macrophages and lymphocytes, and damaged endothelial cells, could secrete IL-6 and chemokines that elicit neutrophils migration into the lungs32,33. Early recruitment of neutrophils was the key to initiating effective host defenses34. IL-6 level in BALF of children with pneumonia was positively correlated with the percentage of neutrophils. On the one hand, IL-6 could promote the survival of neutrophils35, enhance the bactericidal activity of neutrophils, and expand the inflammatory effect to eliminate pathogens36. However, undue release of IL-6 caused excessive inflammatory damage, which in turn leads to lung damage and aggravation of the disease37.
IL-10 is a cytokine with anti-inflammatory effect38. IL-10 could inhibit the effect of activated alveolar macrophages on T helper cells, reduce the release of pro-inflammatory cytokines, and relieve the body’s inflammatory response38. There was a significant positive correlation between IL-6 level and IL-10 level, which may be due to that IL-6 itself can induce IL-10 expression. In this study, IL-10 level in BALF of children with severe pneumonia were significantly increased, suggesting that IL-10 is also one of the indicators for evaluating the severity of pediatric pneumonia. Studies had shown that IL-10 could inhibit the overproduction of neutrophils39. However, in our study, IL-10 level in BALF of children with pneumonia was positively correlated with the percentage of neutrophils. It might be because IL-6 promotes the expression of IL-10 and also promotes the aggregation of neutrophils. So, the level of IL-10, the number and the percentage of neutrophils in BALF can reflect the intensity of the body’s inflammation. Our results also showed that IL-10 level in BALF was negatively correlated with the percentage of macrophages, which might be related to the inhibition of macrophages by IL-1038.
The level of TNF-αwas also positively correlated with the level of IL-6, IL-10, and the percentage of neutrophils. TNF-α seems to have the same effect as IL-6 in amplifying the inflammatory response. TNF-α, secreted by activated alveolar macrophages, is also the major factor affecting the migration of neutrophils to inflammatory sites. Under the synergistic effect of IL-17A and TNF-α, neutrophils migrated rapidly to the inflammation site and persist40. Blocking TNF-α could significantly down-regulate inflammatory41. Like IL-6 and IL-10, TNF-α is also an indicator of the severity of inflammation in the body. TNF-α was a promoter of lung pathological damage42. TNF-α could induce macrophage and neutrophils to release chemokines43. And TNF-α could stimulate the release of platelet activating factors to cause vasodilation, and it could promote the adhesion and migration of leukocytes to inflammatory sites36. It could lead to more severe inflammatory damage in lung tissue. Therefore, the level of TNF-αmay also indicate the severity of pediatric pneumonia.
We speculate that the lungs of children with severe pneumonia are relatively insufficient in anti-inflammatory cytokines, which is well demonstrated in our study by the levels of anti-inflammatory cytokines. The ratios of IL-6 to IL-10 and TNF-α to IL-10 in BALF of children with severe pneumonia were significantly elevated, indicating the excessive release of pro-inflammatory cytokines and the relative insufficiency of anti-inflammatory cytokines in the lungs. The imbalance of pro-inflammatory and anti-inflammatory effects exacerbated lung damage. Other studies supported this finding13,44,45.
In addition, in our study, an increase in the number of inflammatory cells in BALF was also found in children with severe pneumonia. Pro-inflammatory cytokines could help extend the inflammatory response by increasing the total number of inflammatory cells, especially neutrophils46,47. Compared with children with mild pneumonia, the percentage of macrophages in BALF in children with severe pneumonia decreased significantly. This decline was relative, because the number of macrophages in children with severe pneumonia was also increased significantly. While, a decrease in the activity of macrophages was also found in bacteria-infected mice, and this change was closely related to abnormal cytokine signals48. Microbial infection could change the function of macrophages and impaired their phagocytic and killing ability50. In our study, the undue cytokines might inhibit the proliferation and activation of macrophages to avoid more harmful inflammation. Because the levels of cytokines in BALF were negatively correlated with the percentage of macrophages. Both undue accumulation of neutrophils and relative insufficiency of macrophages were associated to lung injury. Unfortunately, due to the lack of data of macrophage activity in BALF in this study, it is not clear whether the decrease in the percentage of macrophages is accompanied by a decrease in macrophage activity.
ROCs of cytokines and inflammatory cells in BALF were used to distinguish severe pneumonia from mild pneumonia. We found that in BALF the levels of IL-6, IL-10 and TNF-α, the ratio of IL-6 to IL-10, the ratio of TNF-α to IL-10, the number of inflammatory cells and neutrophils, the percentage of neutrophils, increased above the threshold could predict severe pediatric pneumonia. And the percentage of macrophage decreased exceeding the threshold could also predict pediatric severe pneumonia. As a result, the signals of inflammatory cytokines and cells in BALF are important biomarkers for predicting the severity of pediatric pneumonia, helping to identify critically ill children more accurately and choose individualized treatment regimens.
And, suppressing of inflammatory storm and regulating of cytokine imbalance should be one of the new therapeutic targets. If the condition of children with pneumonia permits, bronchoalveolar lavage is a promising treatment option to remove and reduce undue cytokines and inflammatory cells for alleviating lung damage. For severe pediatric patients, appropriate anti-inflammatory treatments (including glucocorticoids and immunoglobulins) should be used as an option. Meduri,G.U and colleagues' study51 showed that long-term use of glucocorticoids could reduce systemic inflammatory response in adults patients with ARDS. Local anti-inflammatory treatments are also worth considering. And inhaled hormones may be one of the important anti-inflammatory treatments, because aerosol inhalation of budesonide could effectively prevent the occurrence of pneumonia after thoracotomy in adults52. Of course, the use of glucocorticoids should be more personalized and discreet to achieve greater benefits for children with pneumonia. Other studies reported that intravenous IL-6R antagonists are beneficial for COVID-19 patients53,54, so local anti-cytokine receptor therapy may be applicable for critical pediatric pneumonia.
Our data demonstrated that the cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α, IFN-γ) in BALF of children with pneumonia were positively pairwise correlated. It was also supported by previous studies55–57, showing a cascade of cytokine responses. Moreover, the levels of cytokines (IL-6, IL-10, TNF-α, IFN-γ) in BALF in children with pneumonia were positively correlated with the number of inflammatory cells and the percentage of neutrophils. This indicated that the pro-inflammatory effect of cytokines was associated with the increase of the total number of inflammatory cells in the lungs, especially the accumulation of neutrophils. Previous studies indicated that excessive accumulation and continuous activation of neutrophils could lead to greater cytokine storms and harmful inflammatory responses24,46.
There are some limitations in this study. Previous studies10,11 found that cytokine profiles also played a role in the identifying of pathogens. However, due to individual differences and few children with pneumonia with pathogen mono-infection in our study, no specific profiles of pathogen-related cytokines were found. In addition, because of the ethical restrictions on collecting BALF from healthy children, and it is difficult to determine that relatively healthy children who underwent bronchoscopy and collected BALF had no lung inflammation at all, this study did not set up a healthy child control group.