Complex Immune Dysregulation in COVID-19 Patients with Severe Respiratory Failure

Evangelos J Giamarellos-Bourboulis, Mihai G Netea, Nikoletta Rovina, Karolina Akinosoglou, Anastasia Antoniadou, Nikolaos Antonakos, Georgia Damoraki, Theologia Gkavogianni, Maria-Evangelia Adami, Paraskevi Katsaounou, Maria Ntaganou, Magdalini Kyriakopoulou, George Dimopoulos, Ioannis Koutsodimitropoulos, Dimitrios Velissaris, Panagiotis Koufargyris, Athanassios Karageorgos, Konstantina Katrini, Vasileios Lekakis, Mihaela Lupse, Antigone Kotsaki, George Renieris, Danai Theodoulou, Vassiliki Panou, Evangelia Koukaki, Nikolaos Koulouris, Charalambos Gogos, Antonia Koutsoukou, Evangelos J Giamarellos-Bourboulis, Mihai G Netea, Nikoletta Rovina, Karolina Akinosoglou, Anastasia Antoniadou, Nikolaos Antonakos, Georgia Damoraki, Theologia Gkavogianni, Maria-Evangelia Adami, Paraskevi Katsaounou, Maria Ntaganou, Magdalini Kyriakopoulou, George Dimopoulos, Ioannis Koutsodimitropoulos, Dimitrios Velissaris, Panagiotis Koufargyris, Athanassios Karageorgos, Konstantina Katrini, Vasileios Lekakis, Mihaela Lupse, Antigone Kotsaki, George Renieris, Danai Theodoulou, Vassiliki Panou, Evangelia Koukaki, Nikolaos Koulouris, Charalambos Gogos, Antonia Koutsoukou

Abstract

Proper management of COVID-19 mandates better understanding of disease pathogenesis. The sudden clinical deterioration 7-8 days after initial symptom onset suggests that severe respiratory failure (SRF) in COVID-19 is driven by a unique pattern of immune dysfunction. We studied immune responses of 54 COVID-19 patients, 28 of whom had SRF. All patients with SRF displayed either macrophage activation syndrome (MAS) or very low human leukocyte antigen D related (HLA-DR) expression accompanied by profound depletion of CD4 lymphocytes, CD19 lymphocytes, and natural killer (NK) cells. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production by circulating monocytes was sustained, a pattern distinct from bacterial sepsis or influenza. SARS-CoV-2 patient plasma inhibited HLA-DR expression, and this was partially restored by the IL-6 blocker Tocilizumab; off-label Tocilizumab treatment of patients was accompanied by increase in circulating lymphocytes. Thus, the unique pattern of immune dysregulation in severe COVID-19 is characterized by IL-6-mediated low HLA-DR expression and lymphopenia, associated with sustained cytokine production and hyper-inflammation.

Keywords: COVID-19; HLA-DR; SARS-CoV-2; dysregulation; ferritin; interleukin-6; lymphopenia; macrophage activation; monocytes; respiratory failure.

Conflict of interest statement

Declaration of Interests E.J.G.-B. has received honoraria from AbbVie USA, Abbott CH, InflaRx GmbH, MSD Greece, XBiotech Inc. and Angelini Italy; independent educational grants from AbbVie, Abbott, Astellas Pharma Europe, AxisShield, bioMérieux Inc, InflaRx GmbH, and XBiotech Inc; and funding from the FrameWork 7 program HemoSpec (granted to the National and Kapodistrian University of Athens), the Horizon2020 Marie-Curie Project European Sepsis Academy (granted to the National and Kapodistrian University of Athens), and the Horizon 2020 European Grant ImmunoSep (granted to the Hellenic Institute for the Study of Sepsis). A.A. has received honoraria and independent educational grants from Gilead, Pfizer, MSD, ViiV, Astellas, and Biotest.

Copyright © 2020 Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Characteristics of Immune Dysregulation of COVID-19 (A) Absolute numbers of the molecules of the human leukocyte antigen (mHLA-DR) on CD14 monocytes. Patients with bacterial CAP and CAP caused by SARS-CoV-2 are classified into three states of immune activation: intermediate, immunoparalysis for bacterial CAP and dysregulation for COVID-19, and MAS. (B) Mean fluorescence intensity (MFI) of HLA-DR on CD14 monocytes of healthy volunteers and of patients with CAP caused by SARS-CoV-2 classified according to their state of immune activation. (C) Ferritin concentrations in the serum of patients with bacterial CAP and sepsis and CAP caused by SAR-CoV-2 according to their state of immune activation. (D) Hemophagocytosis score among patients with CAP caused by SARS-CoV-2 classified according to their state of immune activation. (E) Absolute neutrophil counts among patients with CAP caused by SARS-CoV-2 classified according to their state of immune activation. (F) Absolute monocyte counts among patients with CAP caused by SARS-CoV-2 classified according to their state of immune activation. Bars in each graphic represent mean values and standard errors. Statistical comparisons are indicated by the arrows; ns: non-significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Comparisons were done by the Mann-Whitney U test followed by correction for multiple comparisons.
Figure 2
Figure 2
CD4 Cell and NK Cell Cytopenias Are Characteristics of Infection by SARS-CoV-2 (A–E) Absolute counts of CD3+CD4+CD45+ lymphocytes (A), CD3+CD8+CD45+ lymphocytes (B), CD3+CD16+CD56+CD45+ lymphocytes (C), CD3−CD16+CD56+CD45+ cells (D), and CD19+CD45+ lymphocytes (E) among healthy volunteers, patients with CAP caused by the 2009H1N1 influenza virus, and patients with CAP caused by COVID-19. Patients with COVID-19 are classified into three states of immune activation: intermediate, dysregulation, and MAS. (F–J) absolute counts of CD3+CD4+CD45+ lymphocytes (F), CD3+CD8+CD45+ lymphocytes (G), CD3+CD16+CD56+CD45+ lymphocytes (H), CD3−CD16+CD56+CD45+ cells (I), and CD19+CD45+ lymphocytes (J) among patients with severe respiratory failure developing in the field of CAP caused by the 2009H1N1 influenza virus and COVID-19. (K) IL-17 production by PBMCs after stimulation with heat-killed Candida albicans. Bars in each graphic represent mean values and standard errors. Statistical comparisons are indicated by the arrows; ns: non-significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Comparisons were done by the Mann-Whitney U test followed by correction for multiple comparisons.
Figure 3
Figure 3
Moderate Derangement of Circulating Immunoglobulins in Pneumonia Caused by SARS-CoV-2 Serum amounts of IgG subclasses (A–D), IgM (E), and IgA (F) of patients with CAP caused by SARS-CoV-2 are shown. Patients are classified into three states of immune activation: intermediate, dysregulation, and MAS. Findings are compared with those in patients with bacterial CAP, who are classified into three states of immune activation: intermediate, immunoparalysis, and MAS. Bars in each graphic represent mean values and standard errors. Only statistically significant comparisons are indicated by the arrows; ∗p < 0.05; ∗∗p < 0.0001; ∗∗∗p < 0.0001. Comparisons were done by the Mann-Whitney U test followed by correction for multiple comparisons.
Figure 4
Figure 4
Main Features of Immune Dysregulation of Pneumonia Caused by SARS-CoV-2 (A) Production of TNF-α by PBMCs of patients with sepsis caused by bacterial CAP classified into intermediate state of immune activation and immunoparalysis. (B) Production of TNF-α by PBMCs of patients with CAP caused by SARS-CoV-2 classified into three states of immune activation: intermediate, dysregulation, and MAS. (C) Production of TNF-α by PBMCs of patients with SRF developing after infection caused by the 2009H1N1 virus and by SARS-CoV-2. (D) Production of IL-1β by PBMCs of patients with sepsis caused by bacterial CAP classified into intermediate state of immune activation and immunoparalysis. (E) Production of IL-1β by PBMCs of patients with CAP caused by SARS-CoV-2 classified into three states of immune activation: intermediate, dysregulation, and MAS. (F) Production of IL-1β by PBMCs of patients with SRF developing after infection caused by the 2009H1N1 virus and by SARS-CoV-2. (G) Production of IL-6 by PBMCs of patients with sepsis caused by bacterial CAP classified into intermediate state of immune activation and immunoparalysis. (H) Production of IL-6 by PBMCs of patients with CAP caused by SARS-CoV-2 classified into states of immune activation: intermediate, dysregulation and MAS. (I) Production of IL-6 by PBMCs of patients with SRF developing after infection caused by the 2009H1N1 virus and by SARS-CoV-2. (J–L) Serum amounts of TNF-α, IL-6, and CRP of patients with CAP caused by SARS-CoV-2 classified into states of immune activation: intermediate, dysregulation and MAS. Bars in each graphic represent mean values and standard errors. Statistical comparisons are indicated by the arrows; ns: non-significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Comparisons were done by the Mann-Whitney U test followed by correction for multiple comparisons.
Figure 5
Figure 5
Immune Dysregulation Caused by SARS-CoV-2 Is Mediated by IL-6 (A) Negative correlation between serum amounts of IL-6 and the absolute numbers of the mHLA-DR on CD14 monocytes. The Spearman’s (rs) co-efficient of correlation and the respective p value are provided. (B) Correlation between the absolute lymphocyte count and the absolute numbers of mHLA-DR on CD14 monocytes. The rs co-efficient of correlation and the respective p value are provided. (C) Changes of the absolute numbers of mHLA-DR on CD14 monocytes of four patients infected by SARS-CoV-2 with intermediate state of immune activation after incubation with medium and their plasma. (D) Changes of the MFI of HLA-DR on CD14 monocytes of four patients infected by SARS-CoV-2 with intermediate state of immune activation after incubation with medium and their plasma. (E) Changes of the absolute numbers of mHLA-DR on CD14 monocytes of eight patients infected by SARS-CoV-2 with immune dysregulation after incubation with medium and their plasma; modulation by the addition of the specific IL-6 blocker Tocilizumab is also shown. (F) Changes of the MFI of HLA-DR on CD14 monocytes of eight patients infected by SARS-CoV-2 with immune dysregulation after incubation with medium and their plasma; modulation by the addition of the specific IL-6 blocker tocilizumab is also shown. (G) Changes of the absolute lymphocyte count of six patients before and after start of treatment with Tocilizumab. (H) Intracellular staining for IL-6 in CD14 monocytes and in CD4 lymphocytes of three patients infected by SARS-CoV-2 with immune dysregulation. Statistical comparisons are indicated by the arrows; ns: non-significant; ∗p < 0.05; ∗∗p < 0.01.

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