sharon sanders
Editor-in-Chief & President
Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature by monocytes in COVID-19
Abstract
Although alterations in myeloid cells have been observed in COVID-19, the specific underlying mechanisms are not completely understood. Here, we examine the function of classical CD14[SUP]+[/SUP] monocytes in patients with mild and moderate COVID-19 during the acute phase of infection and in healthy individuals. Monocytes from COVID-19 patients display altered expression of cell surface receptors and a dysfunctional metabolic profile that distinguish them from healthy monocytes. Secondary pathogen sensing ex vivo leads to defects in pro-inflammatory cytokine and type-I IFN production in moderate COVID-19 cases, together with defects in glycolysis. COVID-19 monocytes switch their gene expression profile from canonical innate immune to pro-thrombotic signatures and are functionally pro-thrombotic, both at baseline and following ex vivo stimulation with SARS-CoV-2. Transcriptionally, COVID-19 monocytes are characterized by enrichment of pathways involved in hemostasis, immunothrombosis, platelet aggregation and other accessory pathways to platelet activation and clot formation. These results identify a potential mechanism by which monocyte dysfunction may contribute to COVID-19 pathology.
Introduction
COVID-19 is a respiratory tract infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the majority of infections in unvaccinated individuals are mild or asymptomatic, 15% of patients develop moderate to severe disease requiring hospitalisation, and 5% develop critical disease with life-threatening pneumonia, acute respiratory distress syndrome and septic shock[SUP]1[/SUP]. During the acute phase of infection, myeloid cells, including monocytes and macrophages, are the predominant immune cell type in the lungs of COVID-19 patients and play a major role in the pathogenicity of the disease[SUP]2,3[/SUP]. However, contrasting observations have been reported regarding the involvement of myeloid cells in the development of cytokine storms vs. immunosuppression[SUP]4,5[/SUP] and the overactive or deficient type I IFN response generated by myeloid cells in the lungs and in peripheral blood[SUP]6,7,8,9,10,11[/SUP]. Despite these inconsistent reports, most studies have observed dysregulated innate immune responses and reduced expression of human leukocyte antigen DR (HLA-DR) by circulating myeloid cells, which is considered a marker of immune suppression[SUP]10,12,13,14,15,16[/SUP].
Monocytes are blood-circulating, phagocytic, innate immune leukocytes with important functions in pathogen sensing, and innate and adaptive immune response activation during viral infection[SUP]17[/SUP]. Despite their heterogeneity[SUP]18[/SUP], human monocytes are broadly classified into three subsets based on the expression of CD14 and CD16, i.e., classical (CD14[SUP]+[/SUP]CD16[SUP]-[/SUP]), intermediate (CD14[SUP]+[/SUP]CD16[SUP]+[/SUP]), and nonclassical (CD14[SUP]low[/SUP]CD16[SUP]+[/SUP]) monocytes[SUP]17[/SUP]. During viral infection, circulating monocytes infiltrate affected tissues and differentiate into inflammatory macrophages and dendritic cells (DCs)[SUP]19[/SUP], contributing to pathogen clearance and tissue regeneration.
Here, we examine the phenotype and functionality of the main monocyte population in humans, i.e., classical CD14[SUP]+[/SUP] monocytes, in patients with COVID-19 during the acute phase of disease and compare them to those of healthy individuals. We find that ex vivo isolated CD14[SUP]+[/SUP] monocytes from mild and moderate COVID-19 patients are phenotypically different from monocytes from healthy individuals, displaying differential expression of costimulatory and inhibitory receptors, MHC molecules and a dysfunctional metabolic profile that is accompanied by decreased ex vivo NF-κB activation, while maintaining an intact type I IFN antiviral response. Subsequent pathogen sensing ex vivo led to a state of functional unresponsiveness of COVID-19 monocytes that was associated transcriptionally with that of an endotoxin-induced tolerance signature, characterized by the decrease in canonical innate immune functions, including the expression of activation markers and pro-inflammatory cytokine production. In addition, their gene expression signature and function switched from canonical innate immune functions to a pro-thrombotic phenotype characterized by increased expression of pathways involved in immunothrombosis and increased capacity to form cell aggregates with platelets. These results provide a potential mechanism by which innate immune dysfunction in COVID-19 contributes to disease progression.
Results
Phenotypic alterations in COVID-19 monocytes
Global alterations in innate immune cell phenotypes have been identified in severe COVID-19[SUP]11,20,21,22[/SUP]. As the main human monocyte population, we focused on deeply characterizing the ex vivo phenotype of classical CD14[SUP]+[/SUP] monocytes in uninfected healthy individuals and patients with COVID-19 presenting with mild or moderate symptoms (1-2 or 3-4 WHO ordinal scale for COVID-19 severity, respectively) during the acute phase of disease (Dataset 1). The battery of markers examined by high dimensional flow cytometry included MHC molecules and costimulatory and coinhibitory receptors (Fig. 1). Dimensionality reduction tools demonstrated that while some overlap in the global phenotypes was observed among the three study groups, monocytes from healthy individuals were clearly distinct from both mild and moderate COVID-19 on a tSNE plot (Fig. 1a, n = 15 individuals per group). In addition, COVID-19 monocytes could be distinguished based on disease severity, with main cell clusters for both disease severity groups mapping separately on the tSNE plots. Moderate COVID-19 monocytes expressed decreased levels of HLA-DR, in agreement with previous reports[SUP]10,16[/SUP], but in contrast, they displayed increased expression of HLA-ABC compared to both mild disease and uninfected individuals, suggesting a skewed trend towards class I antigen presentation (Fig. 1b, n = 25 healthy, n = 15 mild and n = 17 moderate COVID-19 patients). In addition, moderate COVID-19 monocytes expressed increased levels of the c-type lectin CD301. The decreased expression of the costimulatory receptor CD86 on moderate COVID-19 monocytes compared to healthy and mild COVID-19 patients, the increased expression of the inhibitory receptors TIM-3[SUP]23[/SUP] compared to healthy individuals, and PD1[SUP]24[/SUP] compared to mild COVID-19 monocytes suggest an altered activation profile skewed towards an inhibitory phenotype. In addition, significant differences in the expression of certain markers were found between mild and moderate COVID-19 monocytes. For example, downregulation of HLA-DR and CD86 and upregulation of TIM-3 and HLA-ABC compared to healthy monocytes were only significant in moderate but not in mild COVID-19 monocytes, and the increased expression of CD80 in mild COVID-19 compared to healthy monocytes was not apparent in moderate COVID-19 (Fig. 1b). These results suggest a more profound dysfunction in moderate than in mild COVID-19 monocytes, and they were further confirmed in a second cohort of healthy individuals, mild and moderate COVID-19 patients (Supplementary Fig. 1). Moreover, in agreement with the altered HLA and costimulatory receptor profile of COVID-19 monocytes, we observed an impairment in their capacity to activate SARS-CoV-2-specific CD4[SUP]+[/SUP] and CD8[SUP]+[/SUP] T cells (Supplementary Fig. 2). Thus, CD14[SUP]+[/SUP] monocytes from both mild and moderate COVID-19 patients were able to efficiently activate SARS-CoV-2-specific CD8[SUP]+[/SUP] T cells upon UV-inactivated SARS-CoV-2 stimulation. However, only CD14[SUP]+[/SUP] monocytes from healthy individuals were able to trigger the activation of SARS-CoV-2-specific CD4[SUP]+[/SUP] T cells.
- Allison K. Maher,
- Katie L. Burnham,
- Emma M. Jones,
- Michelle M. H. Tan,
- Rocel C. Saputil,
- Laury Baillon,
- Claudia Selck,
- Nicolas Giang,
- Rafael Argüello,
- Clio Pillay,
- Emma Thorley,
- Charlotte-Eve Short,
- Rachael Quinlan,
- Wendy S. Barclay,
- Nichola Cooper,
- Graham P. Taylor,
- Emma E. Davenport &
- Margarita Dominguez-Villar
Abstract
Although alterations in myeloid cells have been observed in COVID-19, the specific underlying mechanisms are not completely understood. Here, we examine the function of classical CD14[SUP]+[/SUP] monocytes in patients with mild and moderate COVID-19 during the acute phase of infection and in healthy individuals. Monocytes from COVID-19 patients display altered expression of cell surface receptors and a dysfunctional metabolic profile that distinguish them from healthy monocytes. Secondary pathogen sensing ex vivo leads to defects in pro-inflammatory cytokine and type-I IFN production in moderate COVID-19 cases, together with defects in glycolysis. COVID-19 monocytes switch their gene expression profile from canonical innate immune to pro-thrombotic signatures and are functionally pro-thrombotic, both at baseline and following ex vivo stimulation with SARS-CoV-2. Transcriptionally, COVID-19 monocytes are characterized by enrichment of pathways involved in hemostasis, immunothrombosis, platelet aggregation and other accessory pathways to platelet activation and clot formation. These results identify a potential mechanism by which monocyte dysfunction may contribute to COVID-19 pathology.
Introduction
COVID-19 is a respiratory tract infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the majority of infections in unvaccinated individuals are mild or asymptomatic, 15% of patients develop moderate to severe disease requiring hospitalisation, and 5% develop critical disease with life-threatening pneumonia, acute respiratory distress syndrome and septic shock[SUP]1[/SUP]. During the acute phase of infection, myeloid cells, including monocytes and macrophages, are the predominant immune cell type in the lungs of COVID-19 patients and play a major role in the pathogenicity of the disease[SUP]2,3[/SUP]. However, contrasting observations have been reported regarding the involvement of myeloid cells in the development of cytokine storms vs. immunosuppression[SUP]4,5[/SUP] and the overactive or deficient type I IFN response generated by myeloid cells in the lungs and in peripheral blood[SUP]6,7,8,9,10,11[/SUP]. Despite these inconsistent reports, most studies have observed dysregulated innate immune responses and reduced expression of human leukocyte antigen DR (HLA-DR) by circulating myeloid cells, which is considered a marker of immune suppression[SUP]10,12,13,14,15,16[/SUP].
Monocytes are blood-circulating, phagocytic, innate immune leukocytes with important functions in pathogen sensing, and innate and adaptive immune response activation during viral infection[SUP]17[/SUP]. Despite their heterogeneity[SUP]18[/SUP], human monocytes are broadly classified into three subsets based on the expression of CD14 and CD16, i.e., classical (CD14[SUP]+[/SUP]CD16[SUP]-[/SUP]), intermediate (CD14[SUP]+[/SUP]CD16[SUP]+[/SUP]), and nonclassical (CD14[SUP]low[/SUP]CD16[SUP]+[/SUP]) monocytes[SUP]17[/SUP]. During viral infection, circulating monocytes infiltrate affected tissues and differentiate into inflammatory macrophages and dendritic cells (DCs)[SUP]19[/SUP], contributing to pathogen clearance and tissue regeneration.
Here, we examine the phenotype and functionality of the main monocyte population in humans, i.e., classical CD14[SUP]+[/SUP] monocytes, in patients with COVID-19 during the acute phase of disease and compare them to those of healthy individuals. We find that ex vivo isolated CD14[SUP]+[/SUP] monocytes from mild and moderate COVID-19 patients are phenotypically different from monocytes from healthy individuals, displaying differential expression of costimulatory and inhibitory receptors, MHC molecules and a dysfunctional metabolic profile that is accompanied by decreased ex vivo NF-κB activation, while maintaining an intact type I IFN antiviral response. Subsequent pathogen sensing ex vivo led to a state of functional unresponsiveness of COVID-19 monocytes that was associated transcriptionally with that of an endotoxin-induced tolerance signature, characterized by the decrease in canonical innate immune functions, including the expression of activation markers and pro-inflammatory cytokine production. In addition, their gene expression signature and function switched from canonical innate immune functions to a pro-thrombotic phenotype characterized by increased expression of pathways involved in immunothrombosis and increased capacity to form cell aggregates with platelets. These results provide a potential mechanism by which innate immune dysfunction in COVID-19 contributes to disease progression.
Results
Phenotypic alterations in COVID-19 monocytes
Global alterations in innate immune cell phenotypes have been identified in severe COVID-19[SUP]11,20,21,22[/SUP]. As the main human monocyte population, we focused on deeply characterizing the ex vivo phenotype of classical CD14[SUP]+[/SUP] monocytes in uninfected healthy individuals and patients with COVID-19 presenting with mild or moderate symptoms (1-2 or 3-4 WHO ordinal scale for COVID-19 severity, respectively) during the acute phase of disease (Dataset 1). The battery of markers examined by high dimensional flow cytometry included MHC molecules and costimulatory and coinhibitory receptors (Fig. 1). Dimensionality reduction tools demonstrated that while some overlap in the global phenotypes was observed among the three study groups, monocytes from healthy individuals were clearly distinct from both mild and moderate COVID-19 on a tSNE plot (Fig. 1a, n = 15 individuals per group). In addition, COVID-19 monocytes could be distinguished based on disease severity, with main cell clusters for both disease severity groups mapping separately on the tSNE plots. Moderate COVID-19 monocytes expressed decreased levels of HLA-DR, in agreement with previous reports[SUP]10,16[/SUP], but in contrast, they displayed increased expression of HLA-ABC compared to both mild disease and uninfected individuals, suggesting a skewed trend towards class I antigen presentation (Fig. 1b, n = 25 healthy, n = 15 mild and n = 17 moderate COVID-19 patients). In addition, moderate COVID-19 monocytes expressed increased levels of the c-type lectin CD301. The decreased expression of the costimulatory receptor CD86 on moderate COVID-19 monocytes compared to healthy and mild COVID-19 patients, the increased expression of the inhibitory receptors TIM-3[SUP]23[/SUP] compared to healthy individuals, and PD1[SUP]24[/SUP] compared to mild COVID-19 monocytes suggest an altered activation profile skewed towards an inhibitory phenotype. In addition, significant differences in the expression of certain markers were found between mild and moderate COVID-19 monocytes. For example, downregulation of HLA-DR and CD86 and upregulation of TIM-3 and HLA-ABC compared to healthy monocytes were only significant in moderate but not in mild COVID-19 monocytes, and the increased expression of CD80 in mild COVID-19 compared to healthy monocytes was not apparent in moderate COVID-19 (Fig. 1b). These results suggest a more profound dysfunction in moderate than in mild COVID-19 monocytes, and they were further confirmed in a second cohort of healthy individuals, mild and moderate COVID-19 patients (Supplementary Fig. 1). Moreover, in agreement with the altered HLA and costimulatory receptor profile of COVID-19 monocytes, we observed an impairment in their capacity to activate SARS-CoV-2-specific CD4[SUP]+[/SUP] and CD8[SUP]+[/SUP] T cells (Supplementary Fig. 2). Thus, CD14[SUP]+[/SUP] monocytes from both mild and moderate COVID-19 patients were able to efficiently activate SARS-CoV-2-specific CD8[SUP]+[/SUP] T cells upon UV-inactivated SARS-CoV-2 stimulation. However, only CD14[SUP]+[/SUP] monocytes from healthy individuals were able to trigger the activation of SARS-CoV-2-specific CD4[SUP]+[/SUP] T cells.