SARS-CoV-2 infection induces mixed M1/M2 phenotype in circulating monocytes and alterations in both dendritic cell and monocyte subsets

Abstract

Clinical manifestations of SARS-CoV-2 infection range from mild to critically severe. The aim of the study was to highlight the immunological events associated with the severity of SARS-CoV-2 infection, with an emphasis on cells of innate immunity. Thirty COVID-19 patients with mild/moderate symptoms and 27 patients with severe/critically severe symptoms were recruited from the Clinical Center of Kragujevac during April 2020. Flow cytometric analysis was performed to reveal phenotypic and functional alterations of peripheral blood cells and to correlate them with the severity of the disease. In severe cases, the number of T and B lymphocytes, dendritic cells, NK cells, and HLA-DR-expressing cells was drastically decreased. In the monocyte population proportion between certain subsets was disturbed and cells coexpressing markers of M1 and M2 monocytes were found in intermediate and non-classical subsets. In mild cases decline in lymphocyte number was less pronounced and innate immunity was preserved as indicated by an increased number of myeloid and activated dendritic cells, NK cells that expressed activation marker at the same level as in control and by low expression of M2 marker in monocyte population. In patients with severe disease, both innate and adoptive immunity are devastated, while in patients with mild symptoms decline in lymphocyte number is lesser, and the innate immunity is preserved.

Fig 1. Differences in NK cell population dependent on severity of the disease.

(A) Bar chart showing the percentage of NK cells in peripheral blood leukocytes and the proportion of CD57^- and CD57⁺ cells within NK cell population. (B) Gating strategy: lymphocytes were selected using FS/SS properties; NK cells were selected by the absence of CD3 and CD19. (C) NK cells were defined as activated based on the co-expression of CD57.

Fig 2. Decrease of HLA-DR expression is more pronounced in severe cases.

(A) Bar chart showing the percentage of HLA-DR⁺ cells in peripheral blood leukocytes. (B) FSC/SSC dot plot was used to gate leukocytes and to exclude debris. (C) Overlaid histogram presenting HLADR expression in leukocyte population of controls (green), MD (blue) and SD patients (red). (D) Bar chart and representative pseudocolor plots showing percent of HLADR+ and HLADR- cells within monocyte population. (E) Bar chart and representative pseudocolor plots showing percent of HLADR+ and HLADR- cells within B lymphocyte population.

Fig 3. Dendritic cell subsets differ between the patients with mild (MD) and patients with Severe Disease (SD).

Bar charts (left panel) and representative pseudocolor plots (right panel) showing percent of: (A) overall dendritic cells and (B) myeloid DCs, (C) plasmacytoid DCs and (D) activated DCs within dendritic cell population in controls and patients.

Fig 4. Percentage ratio of monocyte subsets is altered in COVID-19 patients.

Gating strategy for identifying monocyte subsets is described in S2 Fig. (A) Bar chart presenting proportion of classical, intermediate and nonclassical monocyte subsets in controls and patients. Representative zebra plots are presenting monocyte subsets in (B) healthy controls, (C) patients with mild (MD) and (D) patients with severe disease (SD).

Fig 5. Co-expression of CD23 and CD38 is the highest in SD patients monocytes.

Monocyte gating strategy is described in S2 Fig. Bar charts (left panel) and representative pseudocolor plots (right panel) are showing percent of (A) CD38+, (B) CD23+ and (C) double positive CD23+CD38+ cells in monocyte population.

Fig 6. Mixed M1/M2 phenotype prevails in intermediate and nonclassical monocyte subsets of SD patients.

Bar charts are showing percent of CD38+, CD23+ and double positive CD23+CD38+ cells in (A) classical, (B) intermediate and (C) nonclassical subsets of monocytes in controls, MD and SD patients.