COVID-19 severity associates with pulmonary redistribution of CD1c+ DCs and inflammatory transitional and nonclassical monocytes

Abstract

SARS-CoV-2 is responsible for the development of coronavirus disease 2019 (COVID-19) in infected individuals, who can either exhibit mild symptoms or progress toward a life-threatening acute respiratory distress syndrome (ARDS). Exacerbated inflammation and dysregulated immune responses involving T and myeloid cells occur in COVID-19 patients with severe clinical progression. However, the differential contribution of specific subsets of dendritic cells and monocytes to ARDS is still poorly understood. In addition, the role of CD8+ T cells present in the lung of COVID-19 patients and relevant for viral control has not been characterized. Here, we have studied the frequencies and activation profiles of dendritic cells and monocytes present in the blood and lung of COVID-19 patients with different clinical severity in comparison with healthy individuals. Furthermore, these subpopulations and their association with antiviral effector CD8+ T cell subsets were also characterized in lung infiltrates from critical COVID-19 patients. Our results indicate that inflammatory transitional and nonclassical monocytes and CD1c+ conventional dendritic cells preferentially migrate from blood to lungs in patients with severe COVID-19. Thus, this study increases the knowledge of specific myeloid subsets involved in the pathogenesis of COVID-19 disease and could be useful for the design of therapeutic strategies for fighting SARS-CoV-2 infection.

Keywords: COVID-19; Dendritic cells; Immunology; Monocytes; T cells.

Figure 1. t-SNE visualization of cell subset distribution in the blood of COVID-19 patients.

(A) t-SNE analysis of myeloid cells from a total of 49 samples (34 COVID-19 patients and 15 non–COVID-19 [NC] controls) gated after exclusion of lineage-positive cells and excluding granulocytes. Upper dot plots on the left show combined density of cell clusters in both patient groups. Lower dot plots on the right display highlighted distribution of each indicated myeloid cell population. Cell populations present in both t-SNE plots are highlighted with a number. Those populations changing in the 2 patient groups are highlighted in red. (B) Quantification of numbered cell populations identified by t-SNE in A from the blood of COVID-19 and NC controls. Statistical significance was calculated using χ² test and FDR multiple-comparison correction. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Analysis of different myeloid subsets in the blood of COVID-19 patients with different clinical severity.

Box-and-whisker plots representing proportions of indicated myeloid cell populations present in the blood of non–COVID-19 (n = 22) control individuals versus either total COVID-19 patients included in the study or patients stratified into groups according to mild (G1; n = 19), severe (SEV, G2; n = 21), and critical (CRIT, G3; n = 24) clinical status as shown in Supplemental Table 1. Median of values is shown. Error bars represent maximum and minimum values. Statistical significance of differences between patient groups was calculated using a Kruskal-Wallis test followed by Dunn′s post hoc test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3. Analysis of associations between Mo subset frequencies and clinical parameters from COVID-19 patients.

Spearman’s correlations between frequencies of C (left), T (center), and NC (right) Mo and values of PCT (upper plots) and CRP (lower plots) detected in the blood of all COVID-19 patients included in the study. P and R values are shown in the upper right corner on each plot.

Figure 4. Activation profiles of myeloid cells from the blood of COVID-19 patients and association with clinical parameters.

(A) Flow cytometry dot plots showing CD40 expression on gated C, T, and NC Mo from representative non–COVID-19 controls and mild (G1), severe (G2), and critical (G3) COVID-19 patients. Fluorescence minus one (FMO) controls (blue) for each cell subset are included for comparison purposes. (B) Box-and-whisker plots representing CD40 MFI on the indicated myeloid cell populations present in the blood of healthy individuals versus either total COVID-19 patients included in the study or patients stratified into groups according to mild (G1; n = 19), severe (G2; n = 21),and critical (G3; n = 24) clinical characteristics specified in Supplemental Table 1. Median is highlighted. Error bars represent maximum and minimum values. Statistical differences between patient groups were calculated using a Kruskal-Wallis test followed by Dunn′s post hoc test for multiple comparisons. TOT, total. *P < 0.05.

Figure 5. Characterization of myeloid cell subsets present in bronchoscopy infiltrates from COVID-19 patients with ARDS.

(A) Box-and-whiskers plots showing percentages of the indicated cell populations in the hematopoietic CD45⁺ infiltrate present in bronchoscopy mucus samples from severe COVID-19 patients (n = 23) presenting ARDS and receiving IMV at ICU. Error bars represent maximum and minim values. Statistical differences between proportions of cell populations within the same infiltrates were calculated using Friedman’s test for multiple comparisons. *P < 0.05; **P < 0.01; ****P < 0.0001. (B and C) Frequencies (B) and CD40 MFI (C) of CD1c⁺ and CD141⁺ cDCs (left) and T and NC Mo (right) in paired blood and bronchoscopy samples from COVID-19 patients presenting with ARDS (n = 15). Statistical significance of differences in frequencies between paired blood vs. bronchoscopy samples (black) or between different cell subsets within either blood (blue) or bronchoscopy infiltrates (pink) was calculated using a 2-tailed matched pairs Wilcoxon’s test. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Box-and-whiskers plots representing comparison of CD40 MFI on the indicated myeloid cell populations present in the bronchoscopy infiltrates of total critical G3 COVID-19 patients. Error bars represent maximum and minimum values. Statistical significance of differences was calculated using Friedman’s test for multiple comparisons. *P < 0.05; **P < 0.01; ****P < 0.0001. (E) Spearman’s correlations between CRP levels in plasma and CD40 MFI on NC Mo present in the bronchoscopy infiltrates of severe COVID-19 patients. Spearman’s P and R values are shown in the upper right corner of the plot.

Figure 6. Association between effector CD8⁺ T cell and inflammatory myeloid cells present in bronchoscopy from COVID-19 patients with ARDS.

(A) Representative flow cytometry analysis of CD38 versus CXCR5 expression on gated CD8⁺ T cells present in the blood (left) and paired bronchoscopy infiltrate (right) from COVID-19 patients with ARDS. Numbers on quadrants represent percentages of positive cells. (B) Box-and-whiskers plots representing analysis of frequencies of CXCR5⁺CD38⁺ (left), CXCR5⁺CD38^– (center), and CXCR5^–CD38⁺ (right) CD8⁺ T cells present in paired blood and bronchoscopy samples from n = 15 COVID-19 patients presenting with ARDS. Frequencies of these CD8⁺ T cell subsets on the blood of n = 17 non–COVID-19 controls were included for reference. Error bars represent maximum and minimum values. Statistical significance of differences in frequencies between paired blood vs. bronchoscopy samples (blue) or comparison with healthy controls (black) was calculated using a 2-tailed matched pairs Wilcoxon’s and Mann-Whitney U test with Bonferroni’s multiple comparison correction, respectively. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Spearman’s and Pearson’s correlations between proportions of the indicated effector CD8⁺ T cells subset and CD40 MFI on T Mo. Spearman’s and Pearson’s P and R values are shown in the upper right corner on each correlation plot.