Deficient Phagocytosis in Circulating Monocytes from Patients with COVID-19-Associated Mucormycosis

Abstract

Cases of rhino-orbital mucormycosis in patients suffering from severe coronavirus disease 2019 (COVID-19) were reported in different parts of the world, especially in India. However, specific immune mechanisms that are linked to susceptibility to COVID-19-associated mucormycosis (CAM) remain largely unexplored. We aimed to explore whether the differential regulation of circulating cytokines in CAM patients had any potential pathogenic links with myeloid phagocyte function and susceptibility to mucormycosis. A small cohort of Indian patients suffering from CAM (N = 9) as well as COVID-19 patients with no evidence of mucormycosis (N = 5) were recruited in the study. Venous blood was collected from the patients as well as from healthy volunteers (N = 8). Peripheral blood mononuclear cells and plasma were isolated. Plasma samples were used to measure a panel of 48 cytokines. CD14⁺ monocytes were isolated and used for a flow cytometric phagocytosis assay as well as a global transcriptome analysis via RNA-sequencing. A multiplex cytokine analysis of the plasma samples revealed reduction in a subset of cytokines in CAM patients, which is known to potentiate the activation, migration, or phagocytic activity of myeloid cells, compared to the COVID-19 patients who did not contract mucormycosis. Compared to monocytes from healthy individuals, peripheral blood CD14⁺ monocytes from CAM patients were significantly deficient in phagocytic function. The monocyte transcriptome also revealed that pathways related to endocytic pathways, phagosome maturation, and the cytoskeletal regulation of phagocytosis were significantly downregulated in CAM patients. Thus, the study reports a significant deficiency in the phagocytic activity of monocytes, which is a critical effector mechanism for the antifungal host defense, in patients with CAM. This result is in concordance with results regarding the specific cytokine signature and monocyte transcriptome. IMPORTANCE A number of cases of mucormycosis, often fatal, were reported among severe COVID-19 patients from India as well as from some other parts of the world. However, specific immunocellular mechanisms that underlie susceptibility to this fungal infection in COVID-19 remain largely unexplored. Our study reports a deficiency in phagocytosis by monocytes in COVID-19 patients who are concomitantly afflicted with mucormycosis, with this deficiency being linked to a characteristic monocyte transcriptome as well as a circulating cytokine signature. The functional phenotype and cytokine signature of the monocytes may provide useful biomarkers for detecting potential susceptibility to mucormycosis in COVID-19 as well as in other viral infections.

Keywords: COVID-19; cytokines; monocytes; mucormycosis; phagocytosis; transcriptome.

FIG 1

Deficient phagocytosis in circulating monocytes in COVID-19 patients with concomitant mucormycosis. (A) Key demographic features, major comorbidities, and relevant pharmacotherapies in the recruited subjects. (B) Heat map showing the clustering of the plasma abundance of 37 cytokines in all subgroups of recruited subjects. Complete linkage clustering was used, utilizing the Pearson distance measurement. (C) Relative plasma abundance of cytokines PDGF-BB, RANTES, and MIF, compared between healthy volunteers, COVID-19 patients without mucormycosis, and COVID-19 patients with concomitant mucormycosis. A two-tailed unpaired t test as well as a one-way analysis of variance (ANOVA) (P < 0.05) was performed. (D) Representative contour plots for the flow cytometric phagocytosis assay from three subgroups. (E) Percent monocytes showing the phagocytosis of the fluorescent latex beads, compared between healthy volunteers, COVID-19 patients without mucormycosis, and COVID-19 patients with concomitant mucormycosis. A two-tailed unpaired t test as well as a one-way analysis of variance (ANOVA) (P < 0.05) was performed. (F) Percent monocytes showing the phagocytosis of the fluorescent Rhizopus spores, compared between healthy volunteers, COVID-19 patients without mucormycosis, and COVID-19 patients with concomitant mucormycosis. A two-tailed unpaired t test as well as a one-way analysis of variance (ANOVA) (P < 0.05) was performed.

FIG 2

The monocyte transcriptomes reveal the downregulation of phagocytosis-related pathways. (A) The dot plot represents the biological processes that are upregulated and downregulated in the COVID-19 with mucormycosis group. The size of the dot represents the number of genes enriched in the processes, and the color of the dot represents the significance (adjusted P value) of the processes. (B) The dot plot represents biological processes that are upregulated and downregulated in the COVID-19 group, compared to those of healthy monocytes. The size of the dot represents the number of genes enriched in the process, and the color of the dot represents the significance (adjusted P value) of the process. (C) The plot represents a selection of differentially expressed genes that are relevant to phagocytic functions in monocytes, compared between different groups of subjects, as indicated.