Innate immune deficiencies are associated with severity and poor prognosis in patients with COVID-19

Abstract

COVID-19 can cause acute respiratory distress syndrome, leading to death in many individuals. Evidence of a deleterious role of the innate immune system is accumulating, but the precise mechanisms involved remain unclear. In this study, we investigated the links between circulating innate phagocytes and severity in COVID-19 patients. We performed in-depth phenotyping of neutrophil and monocyte subpopulations and measured soluble activation markers in plasma. Additionally, anti-microbial functions (phagocytosis, oxidative burst, and NETosis) were evaluated on fresh cells from patients. Neutrophils and monocytes had a strikingly disturbed phenotype, and elevated concentrations of activation markers (calprotectin, myeloperoxidase, and neutrophil extracellular traps) were measured in plasma. Critical patients had increased CD13^low immature neutrophils, LOX-1 + and CCR5 + immunosuppressive neutrophils, and HLA-DR^low downregulated monocytes. Markers of immature and immunosuppressive neutrophils were strongly associated with severity. Moreover, neutrophils and monocytes of critical patients had impaired antimicrobial functions, which correlated with organ dysfunction, severe infections, and mortality. Together, our results strongly argue in favor of a pivotal role of innate immunity in COVID-19 severe infections and pleads for targeted therapeutic options.

Figure 1

Circulating phagocytes are activated in severe COVID-19. (A–D) Expression of activation markers CD66b (A), CD11b (B), CD62L (C), CD16 (D) on neutrophils by flow cytometry. (E–H) Concentration in plasma of myeloperoxidase (MPO, E), lipocalin-2 (F), calprotectin (G) and NETs (H) in COVID-19 patients and controls. (I,J) Surface expression of CD11b (I) and CD14 (J) on monocytes. (K) Soluble CD14 concentration in plasma. (L) Surface expression of HLA-DR on monocytes. Intergroup comparison by Mann–Whitney U test between healthy controls and ICU or non-ICU patients: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, between non-ICU and ICU patients: ^####P < 0.0001, ^###P < 0.001, ^##P < 0.01, ^#P < .05. All boxplots whiskers represent 10th and 90th percentiles. HC healthy controls.

Figure 2

Neutrophil and monocyte subpopulations are disturbed in COVID-19 patients. (A–H) Percentages of positive cells and surface expression of markers related to subpopulations in neutrophils by flow cytometry. (A) LOX-1 + neutrophils, (B), PD-L1 + neutrophils, (C) ILT3 + CD62L + neutrophils, (D) CD13^low neutrophils, (E) CD10 expression on neutrophils, (F) CD11b^lowCD16^low neutrophils, (G) CD49 + neutrophils, (H) HLA-DR expression on neutrophils. (I–K) Percentages of monocytes subpopulations by flow cytometry. (I) CD14 + CD16- classical monocytes, (J) CD14 + CD16 + intermediate monocytes, (K) CD14^low/-CD16 + atypical monocytes. Intergroup comparison by Mann–Whitney U test between healthy controls and ICU or non-ICU patients: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, between non-ICU and ICU patients: ^####P < 0.0001, ^###P < 0.001, ^##P < 0.01, ^#P < .05. All boxplots whiskers represent 10th and 90th percentiles. HC healthy controls.

Figure 3

Association of innate immunity-related markers with severity. (A) Hierarchical clustering of the correlation matrix from phenotypic and soluble markers significantly correlated with SAPS II score or the PaO₂/FiO₂ ratio in ICU patients. Red is for positive correlation and green for negative correlation. Intensity of color is proportional to Spearman correlation coefficient. Cluster A groups markers with a correlation profile similar to SAPS II score. Cluster B groups markers with correlation profiles similar with PaO₂/FiO₂ ratio. (B) Markers significantly different between patient who survived (red) or died (black) during their ICU stay. The histograms represent the median and the error bars the interquartile range. Intergroup comparison by Mann–Whitney U test between survivors and deceased patients, **P < 0.01, *P < 0.05. (C) Hierarchical clustering of the correlation matrix from phenotypic and soluble markers significantly correlated with external oxygen requirement on the day of sampling (O₂), maximal external oxygen requirement (Max_O2) or length of hospitalization in non-ICU patients (stay_lengh). Red is for positive correlation and green for negative correlation. Intensity of color is proportional to Spearman correlation coefficient. Cluster A groups markers with a correlation profile similar to O2, Max_O2 and stay_length. Cluster B groups markers negatively correlated to cluster A. BC_PN: neutrophil absolute count; BC_Mono: monocyte absolute count. Hierarchical clustering was made with Genesis 1.8.1 (Gratz University of Technology, https://genome.tugraz.at/genesisclient/genesisclient_description.shtml).

Figure 4

Anti-microbial functions of circulating phagocytes are impaired in severe COVID-19. (A,B) Measurement of phagocytosis capacity by phRodo-conjugated Zymosan particles uptake in (A) neutrophils and (B) monocytes. (C,D) Measurement of (C) oxidative burst in response to medium, Tumor Necrosis Factor alpha (TNF), lipopolysaccharide (LPS), TLR8 ligand CLO97, and (D) priming of formyl-methionine-leucine-phenylalanine (fMLF)-induced burst by TNF, LPS and CLO-97 in neutrophils. (E) Measurement of neutrophils NETosis capacity in response to medium, phorbol myristate acetate (PMA), peptidoglycan (PGN), TNF and fMLF, LPS and fMLF. Intergroup comparison by Mann–Whitney U test between healthy controls and ICU or non-ICU patients, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05., between non-ICU and ICU patients, ^##P < 0.01, ^#P < .05. All boxplots whiskers represent 10th and 90th percentiles.

Figure 5

Functional impairment of phagocytes is linked to organ dysfunction, septic shock, and mortality in ICU patients. (A,B) Correlation between phagocytosis and SAPS II in (A) neutrophils and (B) monocytes. Rs Spearman correlation coefficient Rho. (C,D) Phagocytosis capacity in patients with (black) or without (red) a septic shock of (C) neutrophils and (D) monocytes. The histograms represent the median and the error bars the interquartile range. Intergroup comparison by Mann–Whitney U test , **P < 0.01, *P < 0.05. (E–F) Phagocytosis capacity in survivors (red) and deceased (black) ICU patients of (E) neutrophils and (F) monocytes. Intergroup comparison by Mann–Whitney U, ***P < 0.001, **P < 0.01.