Interleukin-6 and granulocyte colony-stimulating factor as predictors of the prognosis of influenza-associated pneumonia

Influenza is common detected in respiratory infection that can spread rapidly through communities. Throughout history, influenza pandemics have killed tens of thousands of people [1]. Globally, seasonal influenza causes significant morbidity and mortality. According to the World Health Organization (WHO), 3 to 5 million people suffer from the flu each year, and nearly 10% of the patients die as a result [2]. Pneumonia is an important complication of influenza. Studies have shown that influenza-associated pneumonia is an independent mortality-related factor in cases of influenza [3]. Therefore, the early prediction of the severity of influenza-associated pneumonia is of great significance with regard to reducing the mortality of seasonal influenza. However, biomarkers for disease severity and progress are lacking. The identification of reliable biomarkers of prognosis would help in the prevention and timely treatment of pneumonia in influenza patients.

Cytokines are a diverse group of small proteins that regulate immune and inflammatory responses [4]. A cytokine storm is a highly activated state of systemic immunity that is characterized by excessive or uncontrolled release of proinflammatory cytokines [5]. It had been proved cytokine storms were associated with the severity in many respiratory diseases, such as bronchiolitis and community-acquired pneumonia [6, 7]. The presence of cytokine storms has also been demonstrated in cases of severe influenza, and plays an important role in the influenza severity [8‐10]. Previous studies revealed that excessive cytokines can be detected in influenza patients with poor prognosis [11, 12]. Thus, cytokine levels might be indicative of the prognosis of influenza patients. However, there are very few studies on the correlation between cytokine levels and the prognosis of influenza-associated pneumonia.

In the present study, we have examined the changes in cytokine levels in patients with pneumonia and their association with disease severity. Additionally, and importantly, we have identified biomarkers to predict the prognosis of influenza-associated pneumonia.

This study investigated inflammatory biomarkers that would be useful for predicting the prognosis of patients with influenza who develop pneumonia. Out of 48 cytokines analyzed and compared between pneumonia patients who had a poor prognosis and pneumonia patients who had a good prognosis, the combined predictive value of IL-6 and G-CSF was found to be the most reliable.

In the present study, cough and shortness of breath were the most common symptoms in patients with influenza, and the incidence of dyspnea in patients with pneumonia was significantly higher than that in patients who did not develop pneumonia [13]. These findings are consistent with the results of previous studies (Table 1). Additionally, in the patients with influenza-associated pneumonia, the neutrophil percentage, hypersensitive C-reactive protein level, and lactate dehydrogenase level were significantly higher, while the level of protein was significantly lower, than those in patients without pneumonia (Table 2). This may be caused by the cytokine storms that occur during the course of influenza-associated pneumonia [14, 15]. Fourteen patients with pneumonia were complicated with bacterial or fungal infection. The use of antibiotics, glucocorticoids, and antifungal drugs in patients with influenza-associated pneumonia was significantly higher than that in those without influenza-associated pneumonia. This is probably because patients with pneumonia have more severe disease that necessitates the prevention and control of secondary infections. In our study, the ICU occupancy, mechanical ventilation rate, and mortality in patients with influenza-associated pneumonia patients were significantly higher than those in non-pneumonia patients, and the length of hospital stay of patients with influenza-associated pneumonia was also significantly longer. These findings also corroborate the previous studies [16].

With regard to the cytokine analysis in the present study, we found that the levels of several proinflammatory cytokines (IL-6, IL-18, and IFN-γ), chemotactic proteins (MCP-1/MCP-3 and IL-8), and cell-stimulating factors (G-CSF and M-CSF) were significantly increased in patients with influenza-associated pneumonia (Fig. 1B) Additionally, the neutrophil percentage in patients with influenza-associated pneumonia was significantly higher than that in patients without pneumonia (Table 2) These findings correspond with the immune response pathways that are activated in response to the influenza virus. That is, epithelial cells, lung resident macrophages, and dendritic cells in the lungs produce inflammatory mediators and present the antigen to activate the immune response. Neutrophils are recruited as the first line of defense, and a large number of cytokines are produced to maintain a continuous immune response. Additionally, the bone marrow is mobilized to produce a large number of neutrophils under the stimulation of G-CSF and IL-6 [17, 18]. The neutrophils are activated under the stimulation of IFN-γ [19], and they gather at the inflammatory site under the influence of chemokines (CCL-2/MCP-1, CXCL-8/IL-8, CXCL-9/MIG, and CXCL-10/IP-10) [20, 21]. Thus, the significantly higher levels of cytokines observed in the pneumonia patients in this study may be indicative of a stronger inflammatory immune response.

In our study, the levels of 11 cytokines were found to be significantly elevated in patients with influenza-associated pneumonia. Accordingly, high levels of cytokines have been considered to be related to the high pathogenicity and poor prognosis of influenza for a long time. For example, Ye et al. found that the expression of IL-2, IL-6, IFN-γ, and TNF-α increased significantly in patients with influenza A H1N1 infection [22]. Additionally, Shen et al. found that elevated levels of IL-6, IL-8 and MIP-1 β were associated with a high viral load and poor prognosis in patients with H7N9 influenza infection [23].

In the present study, we found that patients with pneumonia who had a poor prognosis had a significantly higher neutrophil percentage than those who had a good prognosis. Neutrophils can recognize the invasion of pathogens through receptor signaling pathways that include Toll-like receptors, Fc receptors, and G protein-coupled receptors, and release a reticular de-agglutination chromatin, referred to as neutrophil extracellular traps (NETs), that can effectively kill pathogens. However, a couple of studies by Teluguakula et al. have shown that NETs can induce the occurrence of influenza-associated pneumonia [24, 25]. It was reported that a large number of NETs was found in the alveoli, airway, and tissue lesions of patients with influenza-associated pneumonia, and was associated with the occurrence of acute lung injury, acute respiratory distress syndrome, and other complications that led to a poor prognosis [24, 26]. When influenza infection is complicated with bacterial infection, NETs can also promote the production of IFN-γ by dendritic cells under the action of bacterial lipopolysaccharide. In turn, IFN-γ can lead to a poor prognosis by inhibiting bacterial clearance [27]. Accordingly, in our study, we found that the IL-6, M-CSF, G-CSF, and IFN-γ expression levels were positively correlated with the Curb-65 score (Fig. 3). This implies that these cytokines may have a good correlation with the severity of influenza-associated pneumonia.

In this study, we explored the correlation between cytokines. We found that IL-6 was highly correlated to G-CSF in patients with pneumonia (R = 0.613). The correlation was stronger in patients with a poor prognosis (R = 0.740), while it was notably weaker in influenza-associated pneumonia patients with a good prognosis (R = 0.576) (Fig. 3). Similarly, previous studies have also demonstrated that cytokines interact with each other in dynamic ways that involve cytokine receptors and signaling pathways [4]. Additionally, in an inflammatory environment, inflammatory factors, such as antigens and IL-6, can stimulate the production of macrophages, T cells, endothelial cells, and fibroblasts and induce them to secrete G-CSF [28]. Therefore, we speculated that the correlation between IL-6 and G-CSF observed in this study is related to the degree of inflammation.

We further tested whether these cytokines could be used as biomarkers to predict the prognosis of influenza-associated pneumonia. The AUC of ROC for IL-6 was 0.918, and the combination of IL-6 and G-CSF showed a slightly higher value of 0.926. Thus, IL-6 and G-CSF can be used as an excellent combination of biomarkers to predict the prognosis of influenza-related pneumonia (Fig. 4). IL-6 is released by tissue macrophages and is an early and potent inflammatory mediator [29]. In previous studies, IL-6R antagonists were mainly used for the treatment of autoimmune diseases [30‐32], but recently, IL-6R-targeting inhibitors were also found to be effective in the treatment of severe and critical COVID-19 [33]. G-CSF is a classical neutrophil-stimulating activator that plays a role in the specific functional responses of neutrophils to the influenza A virus [34]. Blocking the receptor of G-CSF was found to inhibit edema caused by neutrophils through a reduction in neutrophil recruitment, and did not affect the clearance of pathogens [35]. Based on these findings, the potential benefits of IL-6- and G-CSF-targeted therapy in the treatment of influenza-associated pneumonia must be explored in the future.

This study has limitations. First, there was a lack of description of the cytokine dynamics in patients with influenza so that it is impossible to further verify the correlation between cytokine levels and prognosis in course of the disease. Second, as some of patients had received antiviral or antibiotic therapy before admission, we could not rule out the effect of treatment on cytokines. Third, the number of patients is so small that a logistic regression analysis can not be carried to explore independent factors associated with worse outcomes. Finally, the study was just carried from January 2019 and June 2019 in a single center, the results should be verified by more date.

In summary, in our study, we found that cytokines play an important role in the development of influenza-associated pneumonia. Importantly, our findings showed a significant increase in IL-6 and G-CSF levels in influenza-associated pneumonia patients with a poor prognosis. In the future, the use of IL-6R and G-CSFR antagonists in the treatment of influenza-associated pneumonia patients with high levels of IL-6 and G-CSF needs to be explored.