Neutrophil Extracellular Traps and Neutrophil Oxidized-Modified Proteins in Patients with Pneumonia

 

Background 

The mortality rate of community-acquired pneumonia (CAP) is high in adult patients. The highest mortality has been registered in patients in the older age group with a steady rise in the incidence of morbidity in younger people of working age. 

There is a need to focus research on the role of neutrophils in the pathogenesis of pneumonia as CAP largely determines the outcomes of the same. This research is aimed at evaluating the indices of neutrophils oxidized-modified proteins and oxidative stress in patients with CAP, depending on the severity, and comparison of the frequency of neutrophil extracellular traps responsible for causing the progress of pneumonia. 

Introduction

The development of CAP is diverse among different patients. It is dependent on the etiology and the pathways of penetration of the microorganisms into the lungs. 

The evaluation of the changes in the metabolic and functional status of neutrophils could be one of the urgent areas of research, as their condition is expected to help determine the outcome of acute pneumonia. 

Neutrophils refer to the most abundant group of white cells, the primary function of which is to cause the destruction of bacteria. The activation of neutrophils, secretion of antimicrobial proteins with granules, and the production of reactive oxygen species (ROS) play a key role in eliminating these microorganisms. The long-term activities of granulocytes also make a huge contribution to the damage to pulmonary tissues, resulting in unfavorable outcomes of pneumonia.

Neutrophils perform the protective functions while also being the potential mediators of pulmonary tissue damage. 

Several studies have been conducted to study the indices of oxidative stress to determine the severity of the pathological processes in this regard. However, the number of studies related to oxidative stress of neutrophils in patients with CAP and chronic obstructive pulmonary disease or in patients with CAP based on the severity of the disease is limited. 

Hence, the study of the role of neutrophils and their metabolic and functional status is considered an urgent and promising task. 

To achieve the purpose of this study, oxidative modifications in the neutrophil proteins were evaluated based on the levels of carbonyl derivatives (CD) and the advanced oxidation protein products (AOPP). The activities of the neutrophil enzyme called myeloperoxidase (MPO) and the role of neutrophil extracellular traps were also studied. 

Materials and Methods

This study involves 51 patients diagnosed with CAP. The patients were divided into two groups based on the severity of the pathology.

The first group comprised 32 patients having moderate severity of pneumonia while the second comprised of 19 patients diagnosed with severe pneumonia. The 3rd group - the comparison group – comprised 14 patients with CAP having chronic obstructive pulmonary disease (COPD) while the control group comprised 19 volunteers. 

At the time of this study, nearly 90% of patients refrained from smoking for at least 2 days. 

The eligibility for inclusion in the research included: 

• Age of patients between 18 and 73 years

• Community-acquired pneumonia as confirmed by the laboratory, physical, and instrumental methods

• Secondary home pneumonia on the background of COPD

• A prolonged course of pneumonia (more than 2 weeks) 

• Absence of any acute infectious-inflammatory pathology in other organs 

• Absence of co-existing pathology related to the endocrinal system 

The exclusion criteria included non-infectious infiltrative lung lesions like infarct pneumonia, pulmonary tuberculosis, neoplasms, bronchiectasis, and parasitic invasions.

For the purpose of this study, CAP was diagnosed based on the presence of pulmonary infiltrates, verified X-rays, and clinical pictures (cough with difficult-to-expectorate mucopurulent sputum, shortness of breath, and chest pain). The incidences of tachypnea were recorded in the majority of patients. 

An increase in ESR (erythrocyte sedimentation rate), a shift of leukocyte formula to the left, leukocytosis, and lymphopenia were some abnormal parameters observed. The bacteriological studies of sputum revealed pneumococci as the possible etiological factor responsible for the development of pneumonia in about 79.6 percent of patients.

Statistical Analyses

The evaluation of the parameters between the study groups was performed using the ANOVA (Kruskal–Wallis analysis of variances) and the median tests. The correlation analysis was performed using Spearman correlation to assess the relationships between the levels of AOPP and the activities of MPO.

Results

A statistically significant rise in the level of CD was observed while studying oxidized-modified proteins in neutrophils of patients of all the study groups relative to that of the control group. 

There was no statistically significant difference in the content of CD based on the degrees of CAP severity, and no major difference was observed with the comparison group. An increase in the levels of CD was noted in CAP patients with COPD as compared to the other study groups.

Also, on average, in the group of participants with severe or moderate severity pneumonia, the levels of CD exceeded that of the control group by 89% and 73%, respectively. 

In CAP participants with COPD, the levels of CD were 2.9 times higher than the values in the control group. 

The presence of 2 clusters of CD was detected in all groups of participants with both CAP as well as CAP with COPD. 

The 1st cluster included the value of the CD of neutrophils corresponding to that of the control group. The 2nd cluster included the value of CD of neutrophils exceeding the value in the control group. The study groups were found to differ only in terms of the degree of expression. 

There were no statistically significant differences in the levels of AOPP in the neutrophils between the study groups. It should be noted that in patients with moderate severity or severe pneumonia, there was a tendency for the increase in the AOPP levels, while in CAP patients with COPD, there was a tendency for the reduction relative to that in the control group. 

While studying the activities of neutrophil MPO, there were no significant differences in the study groups. This could be due to the activities of the enzymes that depend on the etiology of the conditions since it has already been shown that the pathogenesis of pneumonia due to influenza infection can reduce the MPO activity. Earlier studies have shown that the increased severity of influenza-associated pneumonia could cause a greater inhibition of the MPO activities of neutrophils. 

Some studies have also suggested that weighting the course of pneumonia could lead to a significant increase in the activities of myeloperoxidase. 

In this study, pneumococci were found to be the prominent etiologic factor responsible for the development of pneumonia. In 20.4% of cases, it was preceded by influenza or acute respiratory viral infections. Hence, the activities of MPO require further research depending on the specific etiology of pneumonia. 

Discussion

The results obtained through this study have revealed a rise in the oxidative stress as well as the intensity of the formation of oxidized-modified proteins, such as AOPP and CD that are the indicators of the free radical production. The development of inflammation and the increased production of ROS by neutrophils along with the imbalances in the antioxidant defense system could trigger the development of oxidative stress.

The oxidative stress, may, in turn, contribute to the damage of pulmonary structures in patients with inflammatory lung diseases, wherein the metabolic activities of neutrophils are not limited only by the oxygen content.

A statistically significant rise in the contents of CD and an increase in the AOPP levels in neutrophils in patients with CAP of moderate as well as severe severities were observed. The phenomenon of the accumulation of modified proteins could be explained by the reduction in the redox potential of cells and the violation of certain processes responsible for their proteolytic degradation. 

In patients with CAP and COPD, the tendency toward a reduction in the contents of AOPP relative to that of the control group with a significant rise in the CD levels was observed. 

Neutrophil extracellular traps were also found in the blood of participants in the study groups. 

In patients with moderate severity of CAP, the number of traps had reached about 31 per 100 neutrophils in about 25% of patients. In patients with severe pneumonia or pneumonia with COPD, the number of patients with NETs was higher, though the number of traps was not more than 8 per 100 neutrophils.

Conclusion

The presence of NETs in the blood of patients could be an unfavorable factor, as the formation of NETs involves the development of the extracellular network-like structures containing histones and DNA. Extracellular histones can induce cell damage and trigger the formation of thrombin. Hence, the results revealed suggest oxidative imbalances in neutrophils that could affect the health of the pulmonary parenchyma and contribute to the worsening of the pathogenesis of this disease.