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

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.

Graphical abstract

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

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

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

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

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 (r_s) 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 r_s 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.