The ELF cytokines IL-5, IL-7, IL-12p70, IL-13, and GM-CSF were undetectable in the ELF of 26 patients (Fig. 1B-1). There were increases in the other cytokines, with median levels of IL-8 at 1045.1 [IQR 178.4–11,688.0] pmol/L and IL-6 at 27.6 [5.2–151.2] pmol/L and measurable levels of IL-17A (0.8 [0.2–2.7] pmol/L), IL-25 (15.3 [0.4–3.9] pmol/L), and IL-31 (42.3 [2.4–85.9] pmol/L). The levels of typical inflammatory cytokines, such as MCP-1, MIP-1β, TNF-α, IL-1β, and IL-10, were not significantly greater in the patients than in healthy individuals.

The cytokines IFN-γ, sCD40L, IL-2, IL-4, IL-17A, IL-17F, IL-23, IL-25, and IL-31 were undetectable in the plasma of the 27 patients, and 26 patients had IL-7, IL-12p70, IL-21, and IL-22 levels that were below the detection limits (Fig. 1B-2). The detected cytokines included MCP-1, MIP-1β, IL-8, and TNF-α, and only the levels of MCP-1 (2.5 [1.1–5.0] pmol/L) and MIP-1β (0.7 [0.6–1.6] pmol/L) were significantly greater in the patients than in healthy volunteers. Low plasma levels of G-CSF (0.3 [< 0.3–0.4] pmol/L), IL-6 (0.2 [0.0–0.6] pmol/L), IL-10 (0.1 [< 0.1–0.2] pmol/L), IL-13 (0.03 [< 0.03–0.03] pmol/L), and IL-33 (0.24 [< 0.24–0.24] pmol/L) were also detected.

The ratios of cytokine concentrations in the ELF to those in the plasma were calculated (Fig. 1C). The ELF/plasma ratio was the highest for IL-8, at a median of 737 [IQR 262–11,688] with a detection frequency (%df) of 96.3%, in 27 patients, with the second highest ratios being those of IL-6 (218 [39–1206], %df = 74.1%), IL-1β (202 [21–6434], 3.7%), IL-31 (169 [9–394], 0.0%), MCP-1 (81 [13–323], 96.3%), MIP-1β (55 [0–1121], 96.3%), and TNF-α (47 [7–1560], 81.5%). These ratios underscore the significant disparity in the cytokine concentrations in the ELF and those in the plasma in COVID-19 patients.

This study assessed 27 COVID-19 patients with AHRF across three pandemic phases in Japan: the 4th wave with the original variant (Mar-Jun 2021), the 5th wave with the Delta variant (Jul–Sep 2021), and the 6th wave with the Omicron variant (Jan-Mar 2022) (Additional file 4: Table S4, Additional file 5: Fig. S1). Notably, the CCI and creatinine levels were lower in the patients of the Delta wave than in those of the 4th wave (Additional file 4: Table S4). The PSI and ferritin levels were greater in the patients of the Omicron wave group than in those of the Delta wave group (Additional file 4: Table S4, Fig. 2B-1).

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]

In a study of 27 patients, subgroups were created based on the PSI and LIV. PSI was categorized as mild (PSI < 90), moderate (90 ≦ PSI < 130), or severe (130 ≦ PSI) (Fig. 2B-2, Additional file 6: Table S5), while LIV was divided into mild (LIV < 40%), moderate (40% ≦ LIV < 50%), or severe (50% ≦ LIV) (Fig. 2A, B-3, Additional file 7: Table S6). The present study revealed that there were more female patients in the moderate PSI group and more older patients in the severe PSI group. Interestingly, the sum of the 25 cytokine concentrations in the ELF and plasma samples did not significantly differ among the PSI groups. The severe LIV subgroup had higher C-reactive protein levels than did the moderate LIV subgroup. Notably, the sum of the 25 cytokine concentrations in the ELF was lower in the severe LIV subgroup than in the moderate group, but no significant difference in plasma cytokine concentrations was observed among the LIV subgroups, indicating that there are different patterns of inflammation based on lung injury severity.