CYTOKINE IMBALANCE IN EXPERIMENTAL BACTERIAL PNEUMONIA

Hospital-acquired infections are one of the most actual problems in global healthcare. Incidences, mortality rates, and treatment costs of these infections continue to escalate, despite the infection control practices and significant advancements in diagnostic and therapeutic technologies

Nosocomial pneumonia ranks third in frequency among hospital-acquired infections. However, in the structure of hospital infection-related mortality, it takes the leading position, accounting for 20% of deaths in patients within intensive care units (ICUs)

The aim of this study was to conduct a comparative analysis of the production of interferon-gamma (IFN-γ) and interleukin-10 (IL-10) during the pneumonia progression induced by Enterobacter spp. and E. coli in an experimental model using laboratory animals.

The study was conducted on 45 white outbred male mice weighing 18–20 grams. The experiment lasted 14 days. The study adhered to the requirements and conditions outlined in the International Guiding Principles for Biomedical Research Involving Animals. The conclusion of the ethical committee for conducting research using laboratory animals has been obtained. The mice once were intranasally infected by strains of Escherichia coli and Enterobacter species isolated from patients with nosocomial pneumonia. A volume of 0.05 ml was administered at a dose corresponding to the LD50 (1×105 CFU/ml for E. coli and 1×103 CFU/ml for Enterobacter spp.). Each group, which was removed from the experiment at the stages of the study, including the control group (intact mice), consisted of 5 animals. Cytokine production was determined by measuring their levels in the blood serum and in the lung homogenate supernatant on days 1, 7, and 14 post-infection, using enzyme-linked immunosorbent assay (ELISA) with reagents of R&D diagnostics Inc. The supernatant (liquid over precipitate) of the lung homogenate was obtained by centrifuging the homogenized lung tissue of mice in 2 ml of physiological solution at 3000 g for 10 minutes. Data analysis was performed using the Wilcoxon signed-rank test and the Mann-Whitney U Test in the SPSS 20.0 statistical software package.

The comparison of cytokine levels in the blood serum and in the lung tissue supernatant over time after contamination revealed unidirectional shifts in both experimental groups. It was expressed as lower systemic and local levels of IFN-γ compared to IL-10. However, the level of cytokines in the serum of mice with pneumonia caused by Enterobacter spp. was more than 20 times higher than in the control group throughout the study. The level of IFN-γ in mice with pneumonia caused by E. coli was little different from the control values, and the level of IL-10 was significantly lower than in intact animals.

Thus, the serum level of cytokines significantly increased in mice infected with Enterobacter spp. in 1 day after the start of the experiment: IFN-γ – up to 119.88±15.32 pg/ml (in the control group – 3.14±0.59 pg/ml), IL-10 – up to 257.19±30.03 pg/ml (in the control group – 15.55±0.83 pg/ml). The cytokine level tended to decrease on day 7, and it statistically significant increase on day 14: IFN-γ – up to 218.58±28.09 pg/ml, IL-10 – up to 356.31±46.48 pg/ml. When mice were infected by E. coli, the level of cytokines in the blood serum decreased in 1 day: IFN-γ – down to 2.85±0.93 pg/ml, IL-10 – down to 11.77±1.79 pg/ml. The level of IL-10 remained at the same level, and IFN-γ increased over time. Monitoring of local cytokines showed that the level of IFN-γ in the group of animals infected with Enterobacter spp. doubled in 1 day when compared with the control group (144.45±21.64 vs 74.33±6.7 pg/ml in the control group). It continued to increase by day 7, then dropped sharply by day 14 of the experiment. The level of IL-10 also significantly increased (377.97±70.76 vs 85.03±5.08 pg/ml in the control group) in this group in 1 day after contamination. The level of IL-10 did not change much by day 7, and it decreased by day 14. The level of local and systemic IFN-γ in mice infected with E. coli decreased in 1 day after contamination (to 56.97±4.96 pg/ml). It increased by day 7 and decreased by day 14. The level of IL-10 almost doubled in the supernatant by day 7 (from 103.61±8.12 to 186.08±5.13 pg/ml) and continued to be high by day 14 after contamination.

The serum levels of opposing cytokines were found significantly lower in experimental pneumonia induced by E. coli than in the supernatant of lung tissue. It indicates the intensity of local inflammation. In contrast, both the supernatant of lung tissue and the serum levels of IFN-γ and IL-10 were significantly elevated in pneumonia caused by Enterobacter spp. It is important to note that the local level of cytokines decreased more than twofold by day 14 post-infection with Enterobacter spp., whereas the concentration of these cytokines increased sharply in the blood serum. The anti-inflammatory IL-10 level significantly increased in the group of mice infected with E. coli.

Differences in the cytokine profile were also observed in the ratio of pro-inflammatory and anti-inflammatory factors using the IFN-γ/IL-10 coefficient. Anti-inflammatory effects predominated in the blood serum of the control group of mice, and the ratio of regulatory cytokines was more balanced in the lung tissue. A significant increase of the regulatory coefficient in the blood serum and its decrease in the supernatants of lung tissue were observed when mice were infected with Enterobacter spp. It indicated the predominance of anti-inflammatory effect in the lung tissue throughout the observation period (Table 1).

Thus, monitoring of enterobacterial pneumonia in the experiment made it possible to detect increased production of IFN-γ and IL-10. The level of IFN-γ differed little from the control values in pneumonia caused by E. coli, and the level of IL-10 was significantly lower than its values in the blood serum of intact animals. The differences in the dynamics of local cytokine production depended on the etiology of pneumonia. Early local activation of IL-10 and conspicuous depression of IFN-γ were observed in pneumonia caused by Enterobacter spp. in 2 weeks.

It indicated the development of a deep defect in cell mediated immunity because early and excessive production of anti-inflammatory cytokines changes mechanisms’ sequence of anti-infectious defense and aggravates the course of the inflammatory process. An increasing local predominance of IL-10 was detected in mice infected with E. coli without a change in the concentration of IFN-γ over time, which indicated inadequate activation of the immune cellular component. Thus, both Enterobacter spp. and E. coli exert a pronounced immunosuppressive effect on the pulmonary defense factors, and it must be considered when selecting methods of pathogenetic therapy for nosocomial pneumonia.