Case study observational research: inflammatory cytokines in the bronchial epithelial lining fluid of COVID-19 patients with acute hypoxemic respiratory failure

Abstract

Background: In this study, the concentrations of inflammatory cytokines were measured in the bronchial epithelial lining fluid (ELF) and plasma in patients with acute hypoxemic respiratory failure (AHRF) secondary to severe coronavirus disease 2019 (COVID-19).

Methods: We comprehensively analyzed the concentrations of 25 cytokines in the ELF and plasma of 27 COVID-19 AHRF patients. ELF was collected using the bronchial microsampling method through an endotracheal tube just after patients were intubated for mechanical ventilation.

Results: Compared with those in healthy volunteers, the concentrations of interleukin (IL)-6 (median 27.6 pmol/L), IL-8 (1045.1 pmol/L), IL-17A (0.8 pmol/L), IL-25 (1.5 pmol/L), and IL-31 (42.3 pmol/L) were significantly greater in the ELF of COVID-19 patients than in that of volunteers. The concentrations of MCP-1 and MIP-1β were significantly greater in the plasma of COVID-19 patients than in that of volunteers. The ELF/plasma ratio of IL-8 was the highest among the 25 cytokines, with a median of 737, and the ELF/plasma ratio of IL-6 (median: 218), IL-1β (202), IL-31 (169), MCP-1 (81), MIP-1β (55), and TNF-α (47) were lower.

Conclusions: The ELF concentrations of IL-6, IL-8, IL-17A, IL-25, and IL-31 were significantly increased in COVID-19 patients. Although high levels of MIP-1 and MIP-1β were also detected in the blood samples collected simultaneously with the ELF samples, the results indicated that lung inflammation was highly compartmentalized. Our study demonstrated that a comprehensive analysis of cytokines in the ELF is a feasible approach for understanding lung inflammation and systemic interactions in patients with severe pneumonia.

Keywords: Bronchial microsampling; COVID-19; Cytokine; Epithelial lining fluid; Multiplex bead-based assay.

Fig. 1

A (1) The microsampling probe (model BC-402C, Olympus Tokyo, Japan) used for collecting bronchial epithelial lining fluid (ELF). (2) The probe tip is comprised of a 2.5-mm outer polyethylene sheath and a 1.9-mm inner polyester fiber rod probe, 20 mm in length, attached to a stainless-steel guide wire. (3) The process of extracting ELF by centrifugation. B The study analyzed cytokine concentrations in COVID-19 patients (normal) with acute hypoxemic respiratory failure (AHRF) compared to healthy volunteers (covid). (1) Cytokine concentrations in the ELF and (2) plasma are presented. C The ratio of bronchial ELF/plasma concentrations. The data included a box plot representing the 25th to 75th percentiles (interquartile range, IQR), the median (centerline), and whisker lines extending to the furthest data points within Q1–1.5 × IQR and Q3 + 1.5 × IQR. Outliers were identified beyond these limits. Significant differences (*p < 0.05) between the normal and COVID-19 patient groups are marked with an asterisk and were assessed using the Kruskal‒Wallis test with Bonferroni correction

Fig. 2

A The pneumonia severity index (PSI), lung infiltration volume (LIV), C-reactive protein (CRP) concentration in the blood, cytokine concentration in the ELF and plasma, provided therapies and comorbidities were recorded for individual patients in order of LIV, including deceased patients (marked by †). B Age, CRP in blood, total cytokine concentration in the ELF and plasma, LIV, and PSI were analyzed. (1) Comparisons among the chronological groups based on when they developed AHRF due to COVID-19. (2) Comparisons among the groups stratified by the severity of pneumonia using the PSI. (3) Comparison among the groups after patients were stratified by the severity of pneumonia using the LIV. The boxplots show the median, individual data points (colored dots), and whisker lines extending to Q1–1.5 × IQR and Q3 + 1.5 × IQR or the last data point within these values. Points outside these limits are considered outliers. Significance (†p < 0.05) was determined using the Kruskal‒Wallis test with Bonferroni correction for multiple comparisons. AHRF Acute hypoxemic respiratory failure, CRP C-reactive protein, ELF Epithelial lining fluid, IQR Interquartile range, LIV Lung infiltration volume (%) [17, 18], PSI Pneumonia severity index [15]