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Id Subject Object Predicate Lexical cue
T1 0-81 Sentence denotes Complex Immune Dysregulation in COVID-19 Patients with Severe Respiratory Failure
T2 83-91 Sentence denotes Abstract
T3 92-99 Sentence denotes Summary
T4 100-184 Sentence denotes Proper management of COVID-19 mandates better understanding of disease pathogenesis.
T5 185-367 Sentence denotes 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.
T6 368-440 Sentence denotes We studied immune responses of 54 COVID-19 patients, 28 of whom had SRF.
T7 441-685 Sentence denotes 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.
T8 686-848 Sentence denotes 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.
T9 849-1068 Sentence denotes 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.
T10 1069-1279 Sentence denotes 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.
T11 1281-1299 Sentence denotes Graphical Abstract
T12 1301-1311 Sentence denotes Highlights
T13 1312-1402 Sentence denotes • Severe COVID-19 patients display immune dysregulation or macrophage activation syndrome
T14 1403-1496 Sentence denotes • Severe respiratory failure is associated with a major decrease of HLA-DR on CD14 monocytes
T15 1497-1570 Sentence denotes • CD4 cell and NK cell cytopenias are characteristics of severe COVID-19
T16 1571-1659 Sentence denotes • IL-6 blocker Tocilizumab partially rescues SARS-CoV-2-associated immune dysregulation
T17 1661-1745 Sentence denotes Proper management of COVID-19 mandates better understanding of disease pathogenesis.
T18 1746-1980 Sentence denotes Giamarellos-Bourboulis et al. describe two main features preceding severe respiratory failure associated with COVID-19: the first is macrophage activation syndrome; the second is defective antigen-presentation driven by interleukin-6.
T19 1981-2061 Sentence denotes An IL-6 blocker partially rescues immune dysregulation in vitro and in patients.
T20 2063-2075 Sentence denotes Introduction
T21 2076-2197 Sentence denotes In December 2019, authorities in Wuhan, China reported a cluster of pneumonia cases caused by an unknown etiologic agent.
T22 2198-2394 Sentence denotes The pathogen was soon identified and sequenced as a novel coronavirus related to the agent of severe acute respiratory syndrome (SARS) and was subsequently termed SARS Coronavirus-19 (SARS-CoV-2).
T23 2395-2522 Sentence denotes The infection spread in the subsequent 3 months on all continents and was declared a pandemic by the World Health Organization.
T24 2523-2689 Sentence denotes As of April 2, 2020, 961,818 documented cases were reported worldwide, and 49,165 patients had died (https://www.who.int/emergencies/diseases/novel-coronavirus-2019).
T25 2690-2784 Sentence denotes This novel coronavirus has a tropism for the lung, causing community-acquired pneumonia (CAP).
T26 2785-2928 Sentence denotes Some patients with pneumonia suddenly deteriorate into severe respiratory failure (SRF) and require intubation and mechanical ventilation (MV).
T27 2929-3013 Sentence denotes The risk of death of these patients is high, reaching even 60% (Arabi et al., 2020).
T28 3014-3086 Sentence denotes Proper management mandates better understanding of disease pathogenesis.
T29 3087-3241 Sentence denotes The majority of physicians use sepsis as a prototype of critical illness for the understanding of severe coronavirus disease 2019 (COVID-19) pathogenesis.
T30 3242-3358 Sentence denotes This is mostly because severe COVID-19 is associated with hyper-cytokinemia (Guan et al., 2020, Huang et al., 2020).
T31 3359-3454 Sentence denotes Lethal sepsis is commonly arising from bacterial CAP, often leading to SRF and the need for MV.
T32 3455-3741 Sentence denotes The peculiar clinical course of CAP caused by SARS-CoV-2, including the sudden deterioration of the clinical condition 7–8 days after the first symptoms, generates the hypothesis that this illness is driven by a unique pattern of immune dysfunction that is likely different from sepsis.
T33 3742-3909 Sentence denotes The features of lymphopenia with hepatic dysfunction and increase of D-dimers (Qin et al., 2020) in these patients with severe disease further support this hypothesis.
T34 3910-4319 Sentence denotes Immune responses of critically ill patients with sepsis can be classified into three patterns: macrophage-activation syndrome (MAS) (Kyriazopoulou et al., 2017), sepsis-induced immunoparalysis characterized by low expression of the human leukocyte antigen D related (HLA-DR) on CD14 monocytes (Lukaszewicz et al., 2009), and an intermediate functional state of the immune system lacking obvious dysregulation.
T35 4320-4418 Sentence denotes We investigated whether this classification might apply to patients with SRF caused by SARS-CoV-2.
T36 4419-4725 Sentence denotes Results revealed that approximately one fourth of patients with SRF have MAS and that most patients suffer from immune dysregulation dominated by low expression of HLA-DR on CD14 monocytes, which is triggered by monocyte hyperactivation, excessive release of interleukin-6 (IL-6), and profound lymphopenia.
T37 4726-4855 Sentence denotes This pattern is distinct from the immunoparalysis state reported in either bacterial sepsis or SRF caused by 2009 H1N1 influenza.
T38 4857-4864 Sentence denotes Results
T39 4866-4964 Sentence denotes All Patients with Severe Respiratory Failure Caused by SARS-CoV-2 Have Immune Dysregulation or MAS
T40 4965-5110 Sentence denotes We assessed the differences of immune activation and dysregulation between SARS-CoV-2 and other known severe infections in three patient cohorts:
T41 5111-5256 Sentence denotes 104 patients with sepsis caused by bacterial CAP; 21 historical patients with 2009 H1N1 influenza; and 54 patients with CAP caused by SARS-CoV-2.
T42 5257-5418 Sentence denotes Patients with bacterial CAP were screened for participation in a large-scale randomized clinical trial with the acronym PROVIDE (ClinicalTrials.gov NCT03332225).
T43 5419-5576 Sentence denotes Patients with 2009 H1N1 influenza have been described in previous publications of our group (Giamarellos-Bourboulis et al., 2009, Raftogiannis et al., 2010).
T44 5577-5690 Sentence denotes The clinical characteristics of patients with bacterial CAP and CAP caused by COVID-19 are described in Table 1 .
T45 5691-5814 Sentence denotes Each cohort (bacterial sepsis and COVID-19) is split into patients who developed SRF and required MV and those who did not.
T46 5815-6536 Sentence denotes Three main features need to be outlined: (1) patients with COVID-19 and SRF are less severe than those with severe bacterial CAP, on the basis of the traditional severity scores of sequential organ failure assessment (SOFA) and acute physiology and chronic health evaluation (APACHE) II; (2) this leads to the conclusion that COVID-19 patients undergo an acute immune dysregulation with deterioration into SRF before the overall state of severity is advanced; and (3) although the burden of co-morbidities of patients with COVID-19, as expressed by the Charlson’s co-morbidity index, is higher among patients with SRF than among patients without SRF, it remains remarkably lower that traditional bacterial CAP and sepsis.
T47 6537-6726 Sentence denotes It was also notable that the admission values of Glasgow Coma Scale scores of patients with bacterial CAP were 8.80 ± 4.76, and that of patients with COVID-19 was 14.71 ± 0.20 (p < 0.0001).
T48 6727-6890 Sentence denotes This finding is fully compatible with clinical descriptions of severe COVID-19: patients are admitted at a relatively good clinical state and suddenly deteriorate.
T49 6891-7017 Sentence denotes Table 1 Baseline Clinical and Laboratory Characteristics of the Cohorts of Bacterial CAP and of Pneumonia Caused by SARS-CoV-2
T50 7018-7067 Sentence denotes No respiratory failure Severe respiratory failure
T51 7068-7125 Sentence denotes Bacterial SARS-CoV-2 p value Bacterial SARS-CoV-2 p value
T52 7126-7156 Sentence denotes Number of patients 48 26 56 28
T53 7157-7245 Sentence denotes Age (years, mean ± SD) 74.8 ± 16.8 59.2 ± 10.3 <0.0001 74.0 ± 12.6∗ 67.8 ± 10.8# <0.0001
T54 7246-7320 Sentence denotes Male gender (n, %) 25 (52.1) 15 (57.7) 0.807 27 (48.2)∗ 25 (89.3)∗∗ 0.0003
T55 7321-7419 Sentence denotes APACHE II score (mean ± SD) 18.50 ± 8.19 5.88 ± 3.40 <0.0001 26.63 ± 8.52∗∗ 10.17 ± 3.64## <0.0001
T56 7420-7511 Sentence denotes SOFA score (mean ± SD) 7.87 ± 3.81 1.50 ± 0.82 <0.0001 11.46 ± 3.15∗∗ 5.71 ± 2.19## <0.0001
T57 7512-7594 Sentence denotes CCI (mean ± SD) 5.53 ± 2.13 2.16 ± 1.46 <0.0001 5.57 ± 2.20∗ 3.39 ± 2.16## <0.0001
T58 7595-7682 Sentence denotes PSI (mean ± SD) 146.5 ± 43.2 80.0 ± 30.7 <0.0001 177.4 ± 40.4∗∗∗ 121.2 ± 28.3## <0.0001
T59 7683-7712 Sentence denotes Laboratory values (mean ± SD)
T60 7713-7838 Sentence denotes  Total white blood cell count (/mm3) 13,852.7 ± 7279.3 6379.6 ± 1993.9 <0.0001 17,666.9 ± 12,799.9∗ 9447.8 ± 3308.6## <0.0001
T61 7839-7944 Sentence denotes  Absolute platelet count (x103 /mm3) 201.3 ± 124.8 243.8 ± 109.1 0.141 224.3 ± 111.0∗ 213.9 ± 71.8∗ 0.654
T62 7945-8011 Sentence denotes  INR 1.28 ± 0.64 1.11 ± 0.15 0.187 1.33 ± 0.45∗ 1.17 ± 0.20∗ 0.077
T63 8012-8092 Sentence denotes  aPTT (secs) 37.19 ± 12.95 33.40 ± 6.22 0.165 38.42 ± 23.00∗ 37.52 ± 9.88∗ 0.844
T64 8093-8182 Sentence denotes  Fibrinogen (mg/dl) 475.6 ± 196.3 528.9 ± 152.5 0.234 495.3 ± 290.5∗ 693.5 ± 188.6# 0.002
T65 8183-8264 Sentence denotes  D-dimers (g/dl) 7.66 ± 13.9 2.76 ± 2.02 0.079 1.46 ± 1.62∗∗ 5.43 ± 6.41∗ <0.0001
T66 8265-8346 Sentence denotes  Creatinine (mg/dl) 1.55 ± 1.00 0.85 ± 0.19 0.001 1.71 ± 0.90∗ 1.11 ± 0.43# 0.001
T67 8347-8433 Sentence denotes  Total bilirubin (mg/dl) 1.43 ± 1.92 0.67 ± 0.50 0.052 1.17 ± 1.88∗ 0.97 ± 0.68∗ 0.588
T68 8434-8510 Sentence denotes  AST (U/l) 155.1 ± 308.1 39.9 ± 28.5 0.062 311.7 ± 748.2∗ 76.6 ± 59.2# 0.102
T69 8511-8587 Sentence denotes  ALT (U/l) 234.6 ± 764.3 40.2 ± 24.9 0.200 175.8 ± 378.0∗ 64.3 ± 62.2∗ 0.126
T70 8588-8613 Sentence denotes Main comorbidities (n, %)
T71 8614-8691 Sentence denotes  Type 2 diabetes mellitus 13 (27.1) 4 (15.4) 0.386 21 (37.5)∗ 6 (21.4)∗ 0.214
T72 8692-8765 Sentence denotes  Chronic heart failure 8 (16.7) 3 (11.5) 0.737 18 (32.1)∗ 4 (14.3)∗ 0.114
T73 8766-8836 Sentence denotes  Coronary heart disease 7 (14.6) 2 (7.7) 0.479 10 (17.9) 5 (17.9)∗ 1.0
T74 8837-8986 Sentence denotes Comparisons with the respective groups without respiratory failure by the Student’s t test: ∗p-non-significant; ∗∗p < 0.05; #p < 0.001; ##p < 0.0001.
T75 8987-9350 Sentence denotes Abbreviations are as follows: ALT, alanine aminotransferase; aPTT, activated partial thromboplastin time; AST, aspartate aminotransferase; APACHE, acute physiology and chronic health evaluation; CCI, Charlson’s comorbidity index; INR, international normalized ratio; PSI, pneumonia severity index; SD, standard deviation; SOFA, sequential organ failure assessment
T76 9351-9701 Sentence denotes Immune classification of patients with SARS-CoV-2 was performed by using the tools suggested for bacterial sepsis, i.e., ferritin more than 4,420 ng/mL for MAS (Kyriazopoulou et al., 2017), and HLA-DR molecules on CD14 monocytes lower than 5,000, in the absence of elevated ferritin, for the immune dysregulation phenotype (Lukaszewicz et al., 2009).
T77 9702-9860 Sentence denotes It was found that contrary to the patients with bacterial CAP and SRF, all patients with SRF and SARS-CoV-2 had either immune dysregulation or MAS (Table 2 ).
T78 9861-10019 Sentence denotes Table 2 Association between Severe Respiratory Failure and Immune Classification among Patients with COVID-19 and Patients with Sepsis Caused by Bacterial CAP
T79 10020-10095 Sentence denotes Number of patients with SRF/total patients [%, (−95% CI, +95% CI)] p value∗
T80 10096-10118 Sentence denotes Bacterial CAP COVID-19
T81 10119-10182 Sentence denotes Intermediate 21/40 [52.5 (27.5-67.1)] 0/26 [0 (0-12.9)] <0.0001
T82 10183-10321 Sentence denotes Immunoparalysis (for bacterial CAP) and immune dysregulation (for COVID-19) or MAS 35/64 [54.7 (42.6-66.3)] 28/28 [100 (87.9-100)] <0.0001
T83 10322-10360 Sentence denotes ∗comparisons by the Fisher exact test.
T84 10361-10413 Sentence denotes Abbreviation is as follows: CI, confidence interval.
T85 10415-10480 Sentence denotes Major Decrease of HLA-DR on CD14 Monocytes Is Associated with SRF
T86 10481-10600 Sentence denotes Immunoparalysis of sepsis is characterized by significant decrease of the number of HLA-DR molecules on CD14 monocytes.
T87 10601-10684 Sentence denotes This also happens in immune dysregulation caused by SARS-CoV-2 (Figures 1A and 1B).
T88 10685-10856 Sentence denotes Although patients with bacterial-CAP-associated MAS also have decreased HLA-DR molecules on CD14 monocytes, their circulating ferritin is significantly higher than normal.
T89 10857-10940 Sentence denotes This is a feature found only in a few patients with SARS-CoV-2 and MAS (Figure 1C).
T90 10941-11083 Sentence denotes The absence of traits of MAS among cases of SARS-CoV-2 with immune dysregulation is further proven by the low scores of hemophagocytosis (HS).
T91 11084-11216 Sentence denotes HScore is proposed as a classification tool for secondary MAS, and values more than 169 are highly diagnostic (Fardet et al., 2014).
T92 11217-11338 Sentence denotes Seven patients with SARS-CoV-2 had HS above this cut-off, and all were properly classified by using ferritin (Figure 1D).
T93 11339-11499 Sentence denotes Among patients with bacterial CAP at an intermediate immune state, the number of molecules of HLA-DR on their CD14 monocytes was lower than in healthy patients.
T94 11500-11688 Sentence denotes However, patients with pneumonia caused by SARS-CoV-2 at an intermediate immune state maintained their number of molecules of HLA-DR on CD14 monocytes much closer to the healthy condition.
T95 11689-11759 Sentence denotes When this number suddenly dropped, SRF supervened (Figures 1A and 1B).
T96 11760-11915 Sentence denotes Moreover, the absolute counts of neutrophils and monocytes were higher among patients with immune dysregulation than patients with MAS (Figures 1E and 1F).
T97 11916-11976 Sentence denotes Figure 1 Characteristics of Immune Dysregulation of COVID-19
T98 11977-12074 Sentence denotes (A) Absolute numbers of the molecules of the human leukocyte antigen (mHLA-DR) on CD14 monocytes.
T99 12075-12275 Sentence denotes 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.
T100 12276-12467 Sentence denotes (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.
T101 12468-12625 Sentence denotes (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.
T102 12626-12755 Sentence denotes (D) Hemophagocytosis score among patients with CAP caused by SARS-CoV-2 classified according to their state of immune activation.
T103 12756-12889 Sentence denotes (E) Absolute neutrophil counts among patients with CAP caused by SARS-CoV-2 classified according to their state of immune activation.
T104 12890-13021 Sentence denotes (F) Absolute monocyte counts among patients with CAP caused by SARS-CoV-2 classified according to their state of immune activation.
T105 13022-13085 Sentence denotes Bars in each graphic represent mean values and standard errors.
T106 13086-13212 Sentence denotes Statistical comparisons are indicated by the arrows; ns: non-significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
T107 13213-13310 Sentence denotes Comparisons were done by the Mann-Whitney U test followed by correction for multiple comparisons.
T108 13312-13390 Sentence denotes CD4 Cell and NK Cell Cytopenias Are Characteristics of Infection by SARS-CoV-2
T109 13391-13633 Sentence denotes The absolute counts of CD3+CD4+CD45+ lymphocytes, CD3+CD8+CD45+ lymphocytes, CD3−CD16+CD56+CD45+ cells, and CD19+CD45+ lymphocytes were lower among patients with COVID-19 compared with those in 10 healthy subjects adjusted for age and gender.
T110 13634-13820 Sentence denotes Compared with patients with CAP caused by 2009H1N1, patients with COVID-19 had lower CD3+CD4+CD45+ lymphocytes but higher CD3+CD16+CD45+ cells and CD19+CD45+ lymphocytes (Figures 2A–2E).
T111 13821-14030 Sentence denotes Those patients with immune dysregulation caused by COVID-19 had lower counts of CD3+ CD4+ CD45+ lymphocytes, CD3+CD8+CD45+ lymphocytes, and CD3−CD16+CD56+CD45+ cells than those at an intermediate immune state.
T112 14031-14315 Sentence denotes When comparisons were limited to patients with SRF and infection by one of the two viruses, it was found that infection by SARS-CoV-2 was accompanied by lower CD3+CD4+CD45+ lymphocytes but with higher CD3−CD16+CD56+CD45+ cells and CD19+CD45+ lymphocytes than 2009H1N1 (Figures 2F–2J).
T113 14316-14446 Sentence denotes The Th17 function as assessed by IL-17 production capacity was downregulated among patients with immune dysregulation (Figure 2K).
T114 14447-14534 Sentence denotes Figure 2 CD4 Cell and NK Cell Cytopenias Are Characteristics of Infection by SARS-CoV-2
T115 14535-14846 Sentence denotes (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.
T116 14847-14962 Sentence denotes Patients with COVID-19 are classified into three states of immune activation: intermediate, dysregulation, and MAS.
T117 14963-15279 Sentence denotes (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.
T118 15280-15362 Sentence denotes (K) IL-17 production by PBMCs after stimulation with heat-killed Candida albicans.
T119 15363-15426 Sentence denotes Bars in each graphic represent mean values and standard errors.
T120 15427-15553 Sentence denotes Statistical comparisons are indicated by the arrows; ns: non-significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
T121 15554-15651 Sentence denotes Comparisons were done by the Mann-Whitney U test followed by correction for multiple comparisons.
T122 15652-15761 Sentence denotes The next question was how this change of CD19+CD45+ lymphocytes is translated to serum immunoglobulins (Igs).
T123 15762-15892 Sentence denotes Concentration of IgGs and their subclasses in the plasma of COVID-19 patients was low, as it was in bacterial CAP (Figures 3A–3D).
T124 15893-15968 Sentence denotes However, IgM and IgA were higher than in bacterial CAP (Figures 3E and 3F).
T125 15969-16161 Sentence denotes Overall, patients at MAS had lower IgG2, IgM, and IgA than those at an intermediate immune state, and patients at immune dysregulation had lower IgM than those at an intermediate immune state.
T126 16162-16256 Sentence denotes Figure 3 Moderate Derangement of Circulating Immunoglobulins in Pneumonia Caused by SARS-CoV-2
T127 16257-16369 Sentence denotes Serum amounts of IgG subclasses (A–D), IgM (E), and IgA (F) of patients with CAP caused by SARS-CoV-2 are shown.
T128 16370-16471 Sentence denotes Patients are classified into three states of immune activation: intermediate, dysregulation, and MAS.
T129 16472-16635 Sentence denotes Findings are compared with those in patients with bacterial CAP, who are classified into three states of immune activation: intermediate, immunoparalysis, and MAS.
T130 16636-16699 Sentence denotes Bars in each graphic represent mean values and standard errors.
T131 16700-16811 Sentence denotes Only statistically significant comparisons are indicated by the arrows; ∗p < 0.05; ∗∗p < 0.0001; ∗∗∗p < 0.0001.
T132 16812-16909 Sentence denotes Comparisons were done by the Mann-Whitney U test followed by correction for multiple comparisons.
T133 16911-17003 Sentence denotes The IL-6 Blocker Tocilizumab Partially Rescues the Immune Dysregulation Driven by SARS-CoV-2
T134 17004-17175 Sentence denotes Sepsis-induced immunoparalysis is characterized by profound deficiency of monocytes for cytokine production upon ex vivo stimulation (Giamarellos-Bourboulis et al., 2011).
T135 17176-17432 Sentence denotes Indeed, production of tumor necrosis factor-α (TNF-α) by LPS-stimulated peripheral blood mononuclear cells (PBMCs) of patients with bacterial CAP classified for immunoparalysis was significantly lower than in patients at an intermediate state (Figure 4 A).
T136 17433-17592 Sentence denotes That was not the case for patients with pneumonia caused by SARS-CoV-2, in whom PBMCs showed sustained TNF-α production after stimulation with LPS (Figure 4B).
T137 17593-17767 Sentence denotes The function of PBMCs in patients with SRF caused by 2009H1N1 was also impaired, and there was lower TNF-α production, a pattern different from COVID-19 patients (Figure 4C).
T138 17768-17924 Sentence denotes Surprisingly, stimulation of IL-1β was lower among patients with immune dysregulation than among patients with an intermediate immune state (Figures 4D–4F).
T139 17925-17998 Sentence denotes IL-6, however, followed the stimulation pattern of TNF-α (Figures 4G–4I).
T140 17999-18297 Sentence denotes This generated the hypothesis that in the case of SRF-aggravated pneumonia caused by SARS-CoV-2, there is a unique combination of defective antigen presentation and lymphopenia that leads to defective function of lymphoid cells, whereas monocytes remain potent for the production of TNF-α and IL-6.
T141 18298-18378 Sentence denotes Figure 4 Main Features of Immune Dysregulation of Pneumonia Caused by SARS-CoV-2
T142 18379-18536 Sentence denotes (A) Production of TNF-α by PBMCs of patients with sepsis caused by bacterial CAP classified into intermediate state of immune activation and immunoparalysis.
T143 18537-18700 Sentence denotes (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.
T144 18701-18829 Sentence denotes (C) Production of TNF-α by PBMCs of patients with SRF developing after infection caused by the 2009H1N1 virus and by SARS-CoV-2.
T145 18830-18987 Sentence denotes (D) Production of IL-1β by PBMCs of patients with sepsis caused by bacterial CAP classified into intermediate state of immune activation and immunoparalysis.
T146 18988-19151 Sentence denotes (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.
T147 19152-19280 Sentence denotes (F) Production of IL-1β by PBMCs of patients with SRF developing after infection caused by the 2009H1N1 virus and by SARS-CoV-2.
T148 19281-19437 Sentence denotes (G) Production of IL-6 by PBMCs of patients with sepsis caused by bacterial CAP classified into intermediate state of immune activation and immunoparalysis.
T149 19438-19593 Sentence denotes (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.
T150 19594-19721 Sentence denotes (I) Production of IL-6 by PBMCs of patients with SRF developing after infection caused by the 2009H1N1 virus and by SARS-CoV-2.
T151 19722-19889 Sentence denotes (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.
T152 19890-19953 Sentence denotes Bars in each graphic represent mean values and standard errors.
T153 19954-20080 Sentence denotes Statistical comparisons are indicated by the arrows; ns: non-significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
T154 20081-20178 Sentence denotes Comparisons were done by the Mann-Whitney U test followed by correction for multiple comparisons.
T155 20179-20336 Sentence denotes As a next step, we measured circulating concentrations of TNF-α, interferon-γ (IFN-γ), IL-6, and C-reactive protein (CRP) in patients infected by SARS-CoV-2.
T156 20337-20479 Sentence denotes IFN-γ was below the limit of detection in all patients (data not shown), indicating that Th1 responses do not contribute to over-inflammation.
T157 20480-20625 Sentence denotes No differences of circulating TNF-α concentrations were found between COVID-19 patients at the three states of immune classification (Figure 4J).
T158 20626-20820 Sentence denotes In contrast, IL-6 and CRP concentrations were significantly higher among patients with immune dysregulation than among patients at an intermediate state of immune activation (Figures 4K and 4L).
T159 20821-21049 Sentence denotes Given that IL-6 was below the limit of detection in some patients with immune dysregulation, we divided them into two groups as follows: seven patients with IL-6 below the limit of detection and 14 patients with detectable IL-6.
T160 21050-21220 Sentence denotes Their severity was similar given that SOFA score and pneumonia severity indexes were similar (p values of comparisons were 0.937 and 0.877, respectively; data not shown).
T161 21221-21411 Sentence denotes IL-6 is known to inhibit HLA-DR expression (Ohno et al., 2016), leading to the hypothesis that IL-6 over-production mediates the low HLA-DR expression on CD14 monocytes of COVID-19 patients.
T162 21412-21731 Sentence denotes In agreement with this, negative correlation was found between serum amounts of IL-6 and the absolute number of HLA-DR molecules on CD14 monocytes of patients with COVID-19 but also between the absolute lymphocyte count and the absolute number of mHLA-DR on CD14 monocytes of patients with COVID-19 (Figures 5A and 5B).
T163 21732-21910 Sentence denotes Furthermore, PBMCs from patients with immune dysregulation were cultured overnight in the presence of plasma of the COVID-19 patients, which was already shown to be rich in IL-6.
T164 21911-22321 Sentence denotes The expression of HLA-DR on CD14 monocytes was strongly inhibited by COVID-19 plasma from patients with immune dysregulation but not by plasma from patients with an intermediate immune state of activation (Figures 5C–5F); the addition of the specific blocker of the IL-6 pathway Tocilizumab partially restored the expression of HLA-DR on monocytes of all patients with immune dysregulation (Figures 5E and 5F).
T165 22322-22466 Sentence denotes Treatment with Tocilizumab in six patients was accompanied by increase of the absolute lymphocyte blood count within the first 24 h (Figure 5G).
T166 22467-22546 Sentence denotes IL-6 was produced partly by CD14 monocytes and partly by CD4 cells (Figure 5H).
T167 22547-22617 Sentence denotes Figure 5 Immune Dysregulation Caused by SARS-CoV-2 Is Mediated by IL-6
T168 22618-22731 Sentence denotes (A) Negative correlation between serum amounts of IL-6 and the absolute numbers of the mHLA-DR on CD14 monocytes.
T169 22732-22820 Sentence denotes The Spearman’s (rs) co-efficient of correlation and the respective p value are provided.
T170 22821-22929 Sentence denotes (B) Correlation between the absolute lymphocyte count and the absolute numbers of mHLA-DR on CD14 monocytes.
T171 22930-23005 Sentence denotes The rs co-efficient of correlation and the respective p value are provided.
T172 23006-23202 Sentence denotes (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.
T173 23203-23385 Sentence denotes (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.
T174 23386-23647 Sentence denotes (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.
T175 23648-23895 Sentence denotes (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.
T176 23896-24010 Sentence denotes (G) Changes of the absolute lymphocyte count of six patients before and after start of treatment with Tocilizumab.
T177 24011-24155 Sentence denotes (H) Intracellular staining for IL-6 in CD14 monocytes and in CD4 lymphocytes of three patients infected by SARS-CoV-2 with immune dysregulation.
T178 24156-24252 Sentence denotes Statistical comparisons are indicated by the arrows; ns: non-significant; ∗p < 0.05; ∗∗p < 0.01.
T179 24254-24264 Sentence denotes Discussion
T180 24265-24600 Sentence denotes All patients with pneumonia caused by SARS-CoV-2 who develop SRF display hyper-inflammatory responses with features of either immune dysregulation or MAS, both of which are characterized by pro-inflammatory cytokines: the immune dysregulation described here, which is driven by IL-6 and not by IL-1β, and MAS, which is driven by IL-1β.
T181 24601-24819 Sentence denotes There are two key features of this immune dysregulation: over-production of pro-inflammatory cytokines by monocytes and dysregulation of lymphocytes characterized by CD4 lymphopenia and subsequently B cell lymphopenia.
T182 24820-24942 Sentence denotes In parallel, the absolute natural killer (NK) cell count is depleted, probably as result of the rapidly propagating virus.
T183 24943-25150 Sentence denotes It was previously shown that the addition of IL-6 in the growth medium of healthy dendritic cells attenuated HLA-DR membrane expression and decreased the production of IFN-γ by CD4 cells (Ohno et al., 2016).
T184 25151-25639 Sentence denotes Three findings of our study are compatible with the proposal that IL-6 is one of the drivers of decreased HLA-DR on the CD14 monocytes: (1) IL-6 concentrations in the blood is inversely associated with HLA-DR expression (Figure 5A), (2) the addition of Tocilizumab in the plasma-enriched medium of cells partially restored the expression of HLA-DR on cell membranes (Figures 5E and 5F), and (3) absolute lymphocyte counts of six patients increased after Tocilizumab treatment (Figure 5G).
T185 25640-25815 Sentence denotes Our findings are indirectly supported by the increase of circulating HLA-DR+ cells during convalescence of one case of COVID-19 of moderate severity (Thevarajan et al., 2020).
T186 25816-26170 Sentence denotes In conclusion, we identified a unique signature of immune dysregulation in the patients with SARS-CoV-2, characterized on the one hand by normal or high cytokine production capacity and increased circulating cytokines (especially IL-6) and on the other hand by defects in the lymphoid function associated with IL-6-mediated decrease in HLA-DR expression.
T187 26171-26460 Sentence denotes These findings support the rationale of launched clinical trials on the efficacy of Anakinra, Sarilumab, Siltuximab, and Tocilizumab (Clinicaltrials.gov NCT04330638, NCT04317092, and NCT04315298 and EudraCT number 2020-001039-29) to elaborate anti-inflammatory responses in these patients.
T188 26462-26474 Sentence denotes STAR★Methods
T189 26476-26495 Sentence denotes Key Resources Table
T190 26496-26533 Sentence denotes REAGENT or RESOURCE SOURCE IDENTIFIER
T191 26534-26544 Sentence denotes Antibodies
T192 26545-26613 Sentence denotes CD14-FITC (RMO52 clone) Beckman Coulter Cat#IM0645U; RRID: AB_130992
T193 26614-26681 Sentence denotes CD45-PC5 (J33 clone) Beckman Coulter Cat#IM2653U; RRID: AB_10641226
T194 26682-26751 Sentence denotes HLA-DR-RD1 (9-49 clone) Beckman Coulter Cat#6604366; RRID: AB_2832962
T195 26752-26837 Sentence denotes IgG1-PE isotype control (679.1Mc7 clone) Beckman Coulter Cat#A07796; RRID: AB_2832963
T196 26838-26925 Sentence denotes IgG1-FITC isotype control (679.1Mc7 clone) Beckman Coulter Cat#A07795; RRID: AB_2832964
T197 26926-27026 Sentence denotes CD3-FITC/CD4-PE Antibody Cocktail (UCHT1/13b8.2 clones) Beckman Coulter Cat#A07733; RRID: AB_2832965
T198 27027-27105 Sentence denotes CD3-FITC/CD8-PE Antibody Cocktail Beckman Coulter Cat#A07734; RRID: AB_2832966
T199 27106-27215 Sentence denotes CD3-FITC/CD(16+56)-PE Antibody Cocktail (UCHT1/3G8/N901 (NKH-1)) Beckman Coulter Cat#A07735; RRID: AB_1575965
T200 27216-27296 Sentence denotes CD19-FITC/CD5-PE Antibody Cocktail Beckman Coulter Cat#IM1346U; RRID: AB_2832967
T201 27297-27389 Sentence denotes Quantibrite Anti-HLA-DR/Anti-Monocyte (L243/MϕP9) BD Biosciences Cat#340827; RRID: AB_400137
T202 27390-27470 Sentence denotes Mouse Anti-Human IL-6-PE (AS12 clone) BD Biosciences Cat#340527; RRID: AB_400442
T203 27471-27516 Sentence denotes Chemicals, Peptides, and Recombinant Proteins
T204 27517-27565 Sentence denotes VersaLyse Lysing Solution Beckman Coulter A09777
T205 27566-27606 Sentence denotes Fixative Solution Beckman Coulter A07800
T206 27607-27670 Sentence denotes eBioscience™ Brefeldin A Solution (1000x) Invitrogen 00-4506-51
T207 27671-27728 Sentence denotes IntraPrep Permeabilization Reagent Beckman Coulter A07802
T208 27729-27787 Sentence denotes RPMI 1640 W/ Stable Glutamine W/ 25 MM HEPES Biowest L0496
T209 27788-27840 Sentence denotes Lymphosep, Lymphocyte Separation Media Biowest L0560
T210 27841-27922 Sentence denotes PBS Dulbecco’s Phosphate Buffered Saline w/o Magnesium, w/o Calcium Biowest L0615
T211 27923-27996 Sentence denotes FBS Superior; standardized Fetal Bovine Serum, EU-approved Biochrom S0615
T212 27997-28050 Sentence denotes Gentamycin Sulfate BioChemica PanReac AppliChem A1492
T213 28051-28113 Sentence denotes Penicillin G Potassium Salt BioChemica PanReac AppliChem A1837
T214 28114-28182 Sentence denotes Lipopolysaccharides from Escherichia coli O55:B5 Sigma-Aldrich L2880
T215 28183-28209 Sentence denotes Critical Commercial Assays
T216 28210-28255 Sentence denotes Human IL-1β uncoated ELISA Invitrogen 88-7261
T217 28256-28301 Sentence denotes Human TNF-α uncoated ELISA Invitrogen 88-7346
T218 28302-28337 Sentence denotes Human IL-6 ELISA Invitrogen 88-7066
T219 28338-28396 Sentence denotes Human IL-17A (homodimer) uncoated ELISA Invitrogen 88-7176
T220 28397-28451 Sentence denotes Human Ferritin ELISA ORGENTEC Diagnostika GmbH ORG 5FE
T221 28452-28490 Sentence denotes Human IFN-γ ELISA Diaclone 950.000.192
T222 28491-28514 Sentence denotes Software and Algorithms
T223 28515-28572 Sentence denotes GraphPad Prism Graphpad Software https://www.graphpad.com
T224 28573-28636 Sentence denotes SPSS IBM https://www.ibm.com/analytics/spss-statistics-software
T225 28638-28659 Sentence denotes Resource Availability
T226 28661-28673 Sentence denotes Lead Contact
T227 28674-28811 Sentence denotes Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Evangelos J.
T228 28812-28858 Sentence denotes Giamarellos-Bourboulis (egiamarel@med.uoa.gr).
T229 28860-28882 Sentence denotes Materials Availability
T230 28883-28931 Sentence denotes This study did not generate new unique reagents.
T231 28933-28959 Sentence denotes Data and Code Availability
T232 28960-29035 Sentence denotes Data of this study are available after communication with the Lead Contact.
T233 29037-29075 Sentence denotes Experimental Model and Subject Details
T234 29076-29221 Sentence denotes Blood was sampled from patients with CAP and sepsis and CAP by SARS-CoV-2 of either gender within the first 24 h of hospital admission (Table 1).
T235 29222-29433 Sentence denotes CAP was defined as the presence of new infiltrate in chest X-ray in a patient without any contact with any healthcare facility the last 90 days; sepsis was defined by the Sepsis-3 criteria (Singer et al., 2016).
T236 29434-29587 Sentence denotes COVID was diagnosed for patients with CAP confirmed by chest X-ray or chest computed tomography and positive molecular testing of respiratory secretions.
T237 29588-29717 Sentence denotes For patients who required MV, blood sampling was performed within the first 24 h from MV and results were used for this analysis.
T238 29718-29938 Sentence denotes Exclusion criteria were infection by the human immunodeficiency virus; neutropenia; and any previous intake of immunosuppressive medication (corticosteroids, anti-cytokine biologicals, and biological response modifiers).
T239 29939-30051 Sentence denotes SRF was defined as severe decrease of the respiratory ratio necessitating intubation and mechanical ventilation.
T240 30052-30326 Sentence denotes The studies were conducted under the 78/17 approval of the National Ethics Committee of Greece; the 23/12.08.2019 approval of the Ethics Committee of Sotiria Athens General Hospital; and the 26.02.2019 approval of the Ethics Committee of ATTIKON University General Hospital.
T241 30327-30444 Sentence denotes Written informed consent was provided by patients or by first-degree relatives in case of patients unable to consent.
T242 30445-30613 Sentence denotes All data of patients with bacterial CAP screened for participation in the randomized clinical trial with the acronym PROVIDE (ClinicalTrials.gov NCT03332225) were used.
T243 30614-30718 Sentence denotes All patients admitted for CAP by SARS-CoV-2 from March 3 until March 30, 2020 participated in the study.
T244 30719-30800 Sentence denotes No randomization to experimental groups was needed according to the study design.
T245 30802-30816 Sentence denotes Method Details
T246 30817-31070 Sentence denotes White blood cells were incubated for 15 min in the dark with the monoclonal antibodies anti-CD14 FITC, anti-HLA-DR-PE, anti-CD45 PC5 (Beckman Coulter, Marseille, France) and Quantibrite HLA-DR/anti-monocyte PerCP-Cy5 (Becton Dickinson, Cockeysville Md).
T247 31071-31398 Sentence denotes White blood cells were also incubated for 15 min with anti-CD3 FITC, anti-CD4 FITC and anti-CD19 FITC (fluorescein isothiocyanate, emission 525 nm, Beckman Coulter); with anti-CD4 PE, anti-CD8 PE, and anti-CD(16+56) PE (phycoerythrin, emission 575nm, Beckman Coulter); and with anti-CD45 PC5 (emission 667 nm, Beckman Coulter).
T248 31399-31482 Sentence denotes Fluorospheres (Beckman Coulter) were used for the determination of absolute counts.
T249 31483-31596 Sentence denotes Cells were analyzed after running through the CYTOMICS FC500 flow cytometer (Beckman Coulter Co, Miami, Florida).
T250 31597-31674 Sentence denotes Isotypic IgG controls stained also with anti-CD45 were used for each patient.
T251 31675-31777 Sentence denotes Results were expressed as absolute molecules of HLA-DR on CD14/CD14+CD45+ cells, percentages, and MFI.
T252 31778-32085 Sentence denotes In separate experiments, whole blood was treated with 10 μg/mL brefeldin A (Invitrogen, Carlsbad, CA, USA) for 60 min at 37°C in 5% CO2 followed by incubation in the dark for 15 min at room temperature with anti-CD4 FITC, anti-CD14 FITC (Beckman Coulter) and anti-CD45 PC5 followed by red blood cells lysis.
T253 32086-32243 Sentence denotes Stained cells were then washed with Dulbecco’s Phosphate Buffered Saline and permeabilized with the IntraPrep Parmeabilization Reagent kit (Beckman Coulter).
T254 32244-32319 Sentence denotes Anti-IL-6 PE (Beckman Coulter) was added and analyzed after flow cytometry.
T255 32320-32469 Sentence denotes Peripheral blood mononuclear cells (PBMCs) were isolated after gradient centrifugation over Ficoll (Biochrom, Berlin, Germany) for 20 min at 1,400 g.
T256 32470-32595 Sentence denotes After three washings in ice-cold PBS pH 7.2, PBMCs were counted in a Neubauer plate with trypan blue exclusion of dead cells.
T257 32596-32812 Sentence denotes They were then diluted in RPMI 1640 enriched with 2mM of L-glutamine, 500 μg/mL of gentamicin, 100 U/mL of penicillin G, 10 mM of pyruvate, 10% fetal bovine serum (Biochrom) and suspended in wells of a 96-well plate.
T258 32813-32885 Sentence denotes The final volume per well was 200 μL with a density of 2 × 106 cells/mL.
T259 32886-32976 Sentence denotes PBMCs were exposed in duplicate for 24 h or 5 days at 37°C in 5% CO2 to different stimuli:
T260 32977-33198 Sentence denotes 10 ng/mL of Escherichia coli O55:B5 lipopolysaccharide (LPS, Sigma, St. Louis, USA); 5 × 105 colony forming units of heat-killed Candida albicans; 25% of patient plasma; or 10 μg/mL of Tocilizumab (Roche, Athens, Greece).
T261 33199-33268 Sentence denotes After incubation, cells were removed and analyzed for flow cytometry.
T262 33269-33448 Sentence denotes Concentrations of TNFα, IL-1β, IL-6, IL-17A and IFNγ were measured in cell supernatants and/or serum in duplicate by an enzyme immunoassay (Invitrogen, Carlsbad, California, USA).
T263 33449-33605 Sentence denotes The lowest detections limits were as follows: for TNF-α, 40 pg/mL; for IL-1β, 10 pg/mL; for IL-6, 10 pg/mL; for IL-17A, 10 pg/mL; and for IFN-γ, 12.5 pg/mL.
T264 33606-33768 Sentence denotes Concentrations of ferritin were measured in serum by an enzyme-immunoassay (ORGENTEC Diagnostika GmbH, Mainz, Germany); the lower limit of detection was 75 ng/mL.
T265 33769-33966 Sentence denotes Six patients with immune dysregulation were treated with single intravenous infusion of 8 mg/kg of Tocilizumab (RoAcremra, Roche) based on the National Health Care off-label provision for COVID-19.
T266 33967-34052 Sentence denotes Absolute lymphocyte blood counts were compared before and after Tocilizumab infusion.
T267 34054-34093 Sentence denotes Quantification and Statistical Analysis
T268 34094-34210 Sentence denotes Qualitative data were presented as percentages and confidence intervals (CIs) and compared by the Fisher exact test.
T269 34211-34419 Sentence denotes Qualitative data were presented as mean and standard deviation and compared by the Student’ s “t-test”; they were presented as mean and standard error and compared by the Mann-Whitney U test for small groups.
T270 34420-34484 Sentence denotes Paired comparisons were done by the Wilcoxon’s rank-signed test.
T271 34485-34545 Sentence denotes Non-parametric correlations were done according to Spearman.
T272 34546-34599 Sentence denotes Any value of p below 0.05 was considered significant.
T273 34601-34616 Sentence denotes Acknowledgments
T274 34617-34771 Sentence denotes The study was funded in part by the 10.13039/100010661Horizon 2020 grant ImmunoSep (847422) and in part by the Hellenic Institute for the Study of Sepsis.
T275 34772-34944 Sentence denotes M.G.N. is supported by an 10.13039/501100000781ERC Advanced Grant (833247) and a Spinoza grant of the 10.13039/501100003246Netherlands Organization for Scientific Research.
T276 34946-34966 Sentence denotes Author Contributions
T277 34967-35112 Sentence denotes E.J.G.-B. conceptualized and designed the study, analyzed the data, wrote the manuscript, and gave approval of the final version to be submitted.
T278 35113-35244 Sentence denotes M.G.N. designed lab experiments, analyzed the data, drafted the manuscript, and gave approval of the final version to be submitted.
T279 35245-35501 Sentence denotes N.R., K.A., A.A., N.A., M.E.A., P.K., M.N., M.K., G.D., I.K., D.V., V.L., M.L., A.K., D.T., V.P., E.K., N.K., and A.K. collected blood samples, collected clinical information, reviewed the manuscript, and gave approval of the final version to be submitted.
T280 35502-35648 Sentence denotes G.D., T.G., P.K., A.K., K.K., and G.R. performed lab experiments, reviewed the manuscript, and gave approval of the final version to be submitted.
T281 35649-35772 Sentence denotes C.G. and A.K. contributed in data analysis, drafted the manuscript, and gave approval of the final version to be submitted.
T282 35774-35798 Sentence denotes Declaration of Interests
T283 35799-36402 Sentence denotes 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).
T284 36403-36520 Sentence denotes A.A. has received honoraria and independent educational grants from Gilead, Pfizer, MSD, ViiV, Astellas, and Biotest.