LTB4-Driven Inflammation and Increased Expression of ALOX5/ ACE2 During Severe COVID-19 in Individuals With Diabetes

Abstract

Diabetes is a known risk factor for severe coronavirus disease 2019 (COVID-19), the disease caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there is a lack of knowledge about the mechanisms involved in the evolution of COVID-19 in individuals with diabetes. We aimed to evaluate whether the chronic low-grade inflammation of diabetes could play a role in the development of severe COVID-19. We collected clinical data and blood samples of patients with and without diabetes hospitalized for COVID-19. Plasma samples were used to measure inflammatory mediators and peripheral blood mononuclear cells, for gene expression analysis of the SARS-CoV-2 main receptor system (ACE2/TMPRSS2), and for the main molecule of the leukotriene B₄ (LTB₄) pathway (ALOX5). We found that diabetes activates the LTB₄ pathway and that during COVID-19 it increases ACE2/TMPRSS2 as well as ALOX5 expression. Diabetes was also associated with COVID-19-related disorders, such as reduced oxygen saturation as measured by pulse oximetry/fraction of inspired oxygen (FiO₂) and arterial partial pressure of oxygen/FiO₂ levels, and increased disease duration. In addition, the expressions of ACE2 and ALOX5 are positively correlated, with increased expression in patients with diabetes and COVID-19 requiring intensive care assistance. We confirmed these molecular results at the protein level, where plasma LTB₄ is significantly increased in individuals with diabetes. In addition, IL-6 serum levels are increased only in individuals with diabetes requiring intensive care assistance. Together, these results indicate that LTB₄ and IL-6 systemic levels, as well as ACE2/ALOX5 blood expression, could be early markers of severe COVID-19 in individuals with diabetes.

Figure 1

Upregulation of LTB₄ signaling in individuals in the DM group. A: Volcano plot with DEGs (blue, upregulated genes; yellow, downregulated genes) in PBMCs from DM patients vs. NDM patients. B: Workflow to identify molecules associated with inflammation and respiratory disorders based on gene expression shown in A and the resulting Venn diagram showing molecules in common among pneumonia, respiratory syndrome, acute lung injury, and inflammation. C: Enriched pathways raised from DEG analyses of PBMCs from DM patients vs. NDM patients, highlighting in red the central position of leukotriene metabolism among pathways. D: Fold change of genes involved with LTB₄ production (ALOX5AP and ALOX5) and signaling (LTB4R) in PBMCs of DM vs. NDM patients. Dotted line = cutoff point for a DEG; solid line = average of the control group. Data are medians. **P < 0.01. Adj., adjusted.

Figure 2

Increased expression of ALOX5 and ACE2/TMPRSS2 receptor system for SARS-CoV-2 infection in individuals with COVID-19 in the DM group. Expressions of ALOX5 (A), ACE2 (B), TMPRSS2 (C), FURIN (D), and CD147 (E) in PBMCs from DM and NDM patients. Data are means. *P < 0.05, **P < 0.01.

Figure 3

ALOX5 expression positively correlates with ACE2 expression in individuals with diabetes and COVID-19, and this is associated with an increased rate in ICU admissions. A: Correlation matrix between ALOX5 and ACE2/TMPRSS2 expression in PBMCs from DM (red) and NDM (gray) patients. B: Correlation analysis between ACE2 and TMPRSS2 expressions in PBMCs of NDM (B) and DM (C) individuals with COVID-19. D: Correlation analysis between ALOX5 and ACE2 expression in PBMCs from DM patients. E to G: Hospitalization type among DM or NDM individuals with COVID-19 based on the expression of ACE2, ALOX5 and TMPRSS2. Data are medians. Spearman r correlation. *P < 0.05, **P < 0.01.

Figure 4

Increased systemic levels of LTB₄ in patients in the DM group, with COVID-19. Levels of LTB4 (A), IL-6 (B), and TNF-α (C) in plasma samples from DM and NDM patients affected by COVID-19. Plasma levels of LTB4 (D), IL-6 (E), and TNF-α (F) in NDM and DM patients with COVID-19 categorized by hospitalization type: clinical beds (CB) or intensive care units (ICU). Data are means. *P < 0.05.

Figure 5

Diabetes induces greater severity of COVID-19. A: Number of days that NDM and DM patients remained hospitalized in CBs or the ICU because of COVID-19. B: Survival curves of NDM and DM patients hospitalized for COVID-19. C: Disease duration measured from the onset of symptoms to hospital discharge for NDM and DM patients with COVID-19. D: O₂ saturation of NDM and DM patients with COVID-19. E: Degree of lung injury in NDM and DM patients with COVID-19. Data are medians in A, B, and D and means in C. *P < 0.05, **P < 0.01.

Figure 6

ALOX5 plays a role in the severity of COVID-19 in individuals with diabetes. A: Correlation matrix among genes, inflammatory parameters, and clinical outcome changes found in all patients with COVID-19. B and C: Dispersion of values with all patients between the correlation of ALOX5 with SpO₂-to-FiO₂ and PaO₂-to-FiO₂ ratios. D: Correlation between oxygen saturation and days of hospitalization. Dotted lines = median of the NDM group. Spearman r correlation. *P < 0.05, **P < 0.01, ****P < 0.0001.