Longitudinal Changes in Peripheral and Alveolar Monocyte and Inflammatory Biomarkers are Distinct in Hypercapnia Patients Following Pulmonary Sepsis-Induced ARDS

Abstract

Background: Hypercapnia, an Acute Respiratory Distress Syndrome (ARDS) complication after pulmonary sepsis, remains enigmatic in terms of its immunological mechanisms. Our study was designed to compare initial values and longitudinal changes in cellular composition and inflammatory biomarkers between pneumonia sepsis-induced ARDS patients without hypercapnia and hypercapnia patients.

Methods: Between Dec 2022-Apr 2023, we prospectively studied 61 severe pneumonia patients. Eleven non-sepsis pneumonia patients were controls; 50 patients with pulmonary sepsis met ARDS criteria, 26 among them developed hypercapnia. We collected clinical data, respiratory parameters, peripheral blood mononuclear cells (PBMCs), and bronchoalveolar lavage fluid (BALF) at Day 1 and Day 7 post-intubation. Single-cell RNA sequencing (ScRNA-seq) was performed between selected hypercapnia and non-hypercapnia patients to characterize immune and cellular profiles. Specimens were analyzed via flow cytometry and cytokine panel.

Results: By compiling clinical data and specimens, we found that hypercapnia patients with ARDS had poorer outcomes and higher mortality. At day 1, ScRNA-seq and cytometric analysis revealed increase in monocytes and activation of cytokine storm genes with elevated interleukin (IL) -1β, IL-12p40, and IL-23 in peripheral blood. In hypercapnia patients, percentage of CD14+CD16- classical monocyte and concentrations of IL-12p40 and IL-23 increased from day 1 to day 7 in both circulation and airways. However, these alterations of cellular phenotype and cytokine decreased during seven-treatment period in non-hypercapnia patients.

Conclusion: We offer novel perspectives on monocyte-centered clusters and associated biomarkers, which play a pivotal role in driving hypercapnia after pulmonary sepsis-induced ARDS. Our study provides fresh insights into the immunological mechanisms underlying hypercapnia in ARDS, laying the foundation for useful therapeutic targets to improve patient outcomes.

Keywords: chemokine; cytokine storm; hypercapnia; monocyte; pulmonary sepsis-induced ARDS.

Figure 1

Alterations in PaCO₂ correlate with disease severity, ventilator status and higher mortality. Within the initial 24 hours of mechanical ventilation, demographic and clinical features were documented for patients with non-sepsis pneumonia (controls, n=11) and pulmonary sepsis-induced ARDS with hypercapnia (n=26) and pulmonary sepsis-induced ARDS with non-hypercapnia (controls, n=24). (A and D) Hypercapnic patients exhibited significantly increased severity scores and higher 28-day mortality rates compared to the non-hypercapnic controls. (B) Respiratory variables in blood gas were correlated with PaCO2 changes despite similar baseline PaO2:FIO2 ratios. (C) On intubation day, pulmonary sepsis-induced ARDS with hypercapnia patients showed decreased respiratory compliance with elevated driving pressure (DP) compared to the non-hypercapnic controls. Mean values with standard errors are represented. A p-value of <0.05 was considered statistically significant. Statistical comparisons are indicated by arrows: *P < 0.05 comparing non-sepsis pneumonia vs pulmonary sepsis-induced ARDS groups, **P < 0.05 comparing pulmonary sepsis-induced ARDS without hypercapnic vs with hypercapnic groups.

Figure 2

ScRNA-seq analysis of PBMCs in Pulmonary Sepsis-Induced ARDS reveals altered cell type composition and monocyte subset accumulation in hypercapnia group, confirmed by flow cytometry. The ScRNA-seq analysis of PBMCs from pulmonary sepsis-induced ARDS patients (one from non-hypercapnia group, three from hypercapnia group) revealed distinct cell type changes in hypercapnia cases. Subsequently, CD14 and CD16 were utilized to identify percentages of CM and IM via flow cytometry in non-hypercapnia (n = 24) and hypercapnia (n = 26) patients. (A) Uniform Manifold Approximation and Projection (UMAP) analysis identified six major cell clusters, with altered proportions of neutrophils, T cells and monocytes in hypercapnia patients. (B) Monocyte phenotypes were annotated based on CD14 and CD16 expression. UMAP analysis identified altered proportions of intermediate monocytes (CD14+CD16+,IM) and classical monocytes (CD14+CD16−,CM) subsets in hypercapnia patients. (C) Flow cytometry analysis confirmed accumulation of CM and IM monocyte subsets in peripheral blood of hypercapnia patients, from initial day to day 7. The values presented represent the means ±SEM. Differences between groups were statistically analyzed using one-way ANOVA. Significant differences were indicated as: *P < 0.05 when comparing pulmonary sepsis-induced ARDS without hypercapnic vs with hypercapnic groups at same time point, and **P < 0.05 when comparing initial day with day 7 within same group.

Figure 3

Gene set analysis of hypercapnia monocytes reveals cytokine storm signatures, convinced by increased cytokines and persistent IL-12p40 and IL-23 elevation during disease period. (A) Genes associated with cytokine storm upregulated in peripheral blood of hypercapnia patients. Heatmap showing expression levels and weights assigned to each of the 8 genes related to cytokines and chemokines that distinguish between pulmonary sepsis-induced ARDS patients with hypercapnia (n = 3) and those without hypercapnia (n = 1). (B) Functional enrichment analysis identified distinct gene signatures related to pathways of inflammatory cytokines and chemokines activation in hypercapnia patients. (C) Peripheral blood supernatants revealed elevated cytokines, including serum IL-1β, IL-RA, IP10, TARC, IL-12p40, IL-23, and IL-5, in hypercapnia patients within 48 hours after incubation. (D) IL-12p40 and IL-23 levels were significantly higher in hypercapnia than non-hypercapnia controls both at early and late stages of ARDS, and persistent IL-12p40 and IL-23 elevation during study period. (A and B) Each bubble plot graphically represents genes differentially expressed in hypercapnia samples compared to non-hypercapnia controls, with colors indicating the contributions of individual hypercapnia samples. All P values comparing non-hypercapnia vs hypercapnia for density plots were significantly low, all below 10E-50. (C and D) Mean values with standard errors are represented. A p-value of <0.05 was considered statistically significant. (C) Significant differences were indicated as: *P < 0.05 comparing non-sepsis pneumonia vs pulmonary sepsis-induced ARDS groups, **P < 0.05 comparing pulmonary sepsis-induced ARDS without hypercapnic vs with hypercapnic groups. (D) Significant differences were indicated as: *P < 0.05 when comparing pulmonary sepsis-induced ARDS without hypercapnic vs with hypercapnic groups at same point, and **P < 0.05 when comparing initial day with day 7 within same group.

Figure 4

Similar kinetics of cellular composition and activated cytokines storm in BALF from matched hypercapnia patients. Cells and cytokines from bronchoalveolar lavage fluid (BALF) of non-sepsis pneumonia (n=11), pulmonary sepsis-induced ARDS with non-hypercapnia (n=24), and pulmonary sepsis-induced ARDS with hypercapnia (n=26) patients were isolated and analyzed. (A) In the airway, CM and IM monocyte subsets showed similar kinetics to those in circulation, with increased percentages in hypercapnia than non-hypercapnia from initial day to day 7. (B) Kinetic cytometry revealed elevation of CD14+CD206+ monocyte-derived alveolar macrophages at day 7 in hypercapnia patients. (C) BALF supernatants showed similar cytokine elevations in hypercapnia patients, including TNF-α and IL-10. All values are presented as means ± SEMs. Statistical differences between groups were evaluated using one-way ANOVA. (A and B) Significant differences were indicated as: *P < 0.05 when comparing non-hypercapnia with hypercapnia groups at same point, and **P < 0.05 when comparing initial day with day 7 within same group. (C) *P < 0.05 comparing non-sepsis pneumonia vs pulmonary sepsis-induced ARDS groups, **P < 0.05 comparing pulmonary sepsis-induced ARDS without hypercapnic vs with hypercapnic groups.