Abstract
COVID19 pandemic has so far caused over three hundred thousand deaths worldwide, primarily due to complications from SARS-CoV-2-associated acute respiratory distress syndrome (ARDS). While an ARDS-driven hyperinflammatory phenotype is associated with higher mortality in non-COVID patients, there is little information on how cytokines and chemokines expressions correlate with clinical outcomes in COVID19 patients. We prospectively enrolled a cohort of 41 patients with acute respiratory distress syndrome on mechanical ventilation. Patients’ blood was obtained at enrollment and outcome measures were liberation from mechanical ventilation and hospital-free days. We determined the expression levels of 44 circulating cytokines/chemokines and found 13 of them associated with worse outcomes. After correcting for multiple comparisons/false discovery rate, only one chemokine (CCL19) remained significantly associated with outcomes (p=0.009). Although not described in association with COVID19, this chemokine was previously found elevated in an animal model of SARS-CoV. Moreover, CCL19 seems to be relevant for bronchus-associated lymphoid tissue (BALT) maintenance and for lung immunity to influenza virus. While this finding requires corroboration, CCL19 determination could facilitate early identification of COVID19-ARDS patients at higher risk of death and be novel target for immunotherapy in this setting.
Introduction:
COVID19 pandemic has so far caused over three hundred thousand deaths worldwide, primarily due to complications from SARS-CoV-2-associated acute respiratory distress syndrome (ARDS)1. While an ARDS-driven hyperinflammatory phenotype is associated with higher mortality in non-COVID patients2, there is little information on how cytokines and chemokines expressions correlate with clinical outcomes in COVID19 patients1. Indeed, emerging evidence suggests that there is a novel and complex inflammatory signature in COVID19-assoicated ARDS that is not found in other patients with ARDS3. While the general cytokine profile of COVID19 patients was recently reported4, there is no data about the association of circulating cytokine expression with ICU outcomes. In this study, we interrogated the cytokine/chemokine profile of COVID19-driven ARDS patients and hypothesized that a unique circulating signature would be associated with worse outcomes.
Methods:
We conducted a prospective cohort study involving 41 patients with diagnoses of COVID19 who required mechanical ventilation due to ARDS and were admitted to the Intensive Care Unit (ICU) of Albany Medical Center in New York between March 20 and April 17, 2020. Ethical approval was obtained from Albany Medical College Committee on Research Involving Human Subjects (IRB# 5670-20). Patients were considered for enrollment if they were older than 18 years and were admitted to the ICU for invasive mechanical ventilation due to COVID19-associated respiratory distress. Exclusion criteria were imminent death or inability to provide consent, which was obtained from the patient or a legally authorized representative. The average age of the cohort was 59.9 years (IQR 50-71.5) and there was a significant male predominance (68.3%). Other patients baseline characteristics are presented in Table 1. At the time of enrollment, blood samples were obtained and immediately centrifuged for plasma fractionation. Samples were frozen for later cytokines/chemokines determinations using a Human XL Cytokine magnetic Luminex-Discovery Fixed Panel (R&D Systems #LKTM014) performed simultaneously in duplicates within a week of enrollment completion. The primary outcome variable was patients’ liberation from mechanical ventilation at day 28 after enrollment. Patients who died or remained ventilated longer than 28 days were grouped together (group 1, worse outcome) and compared with the rest of the cohort (group 2, better outcome); secondary outcome measure was hospital-free days at day 30 post enrollment.
Table 1: Baseline characteristics of the cohort of.
Patients baseline characteristics in the whole cohort. Comparison of clinical and laboratory variables between patients in the group with better and worse outcomes, defined as ability to be liberated from mechanical ventilation at day 28 after enrollment. Data are presented as median with interquartile range (IQR); unless otherwise indicated. APACHE II: Acute Physiology and Chronic Health Evaluation II; SAPS II: Simplified Acute Physiology Score; SOFA: Sequential Organ Failure Assessment. ECMO: Extracorporeal membrane oxygenation; CRP: C-reactive protein. Significant p values are indicated in bold font.
| Variables | Entire cohort | >0 Ventilatory-free days Better outcome |
0 Ventilatory-free days Worse outcome |
p Value |
|---|---|---|---|---|
| Sex- N (%) | n=41 | n=22 | n=19 | |
| Male | 28 (68.3%) | 15 (68.2%) | 13 (68.4%) | 0.98 |
| Female | 13 (31.7%) | 7 (31.8%) | 6 (31.6%) | 0.98 |
| Age (IQR) | ||||
| 59.9 (50-71.5) | 58.7 (53-67) | 61.2 (46-73) | 0.58 | |
| Ethnicity- N (%) | ||||
| White | 11 (26.8%) | 7 (31.8%) | 4 (21.1%) | 0.44 |
| African American | 5 (12.2%) | 1 (4.5%) | 4 (21.1%) | 0.11 |
| Asian | 2 (5.9%) | 1 (4.5%) | 1 (5.3%) | 0.91 |
| Hispanic | 13 (31.7%) | 5 (22.7%) | 8 (42.1%) | 0.18 |
| Other | 10 (24.4%) | 8 (36.4%) | 2 (10.5%) | 0.06 |
| Comorbidities- N(%) | ||||
| Congestive heart failure | 1 (2.4%) | 1 (4.5%) | 0 (0%) | 0.35 |
| Pulmonary disease | 6 (14.6%) | 5 (22.7%) | 1 (5.3%) | 0.11 |
| Diabetes Mellitus | 16 (39.1%) | 9 (40.9%) | 7 (36.8%) | 0.79 |
| renal Disease | 4 (9.8%) | 1 (4.5%) | 3 (15.8%) | 0.23 |
| Cancer | 2 (4.9%) | 1 (4.5%) | 1 (5.3%) | 0.91 |
| HIV/AIDS | 1 (2.4%) | 1 (4.5%) | 0 (0%) | 0.35 |
| Charlson score index | 3.05 (1-4) | 3.09 (2-3.25) | 3.00 (1-5) | 0.90 |
| Severity scores (IQR) | ||||
| APACHEII | 24.17 (18.5-28.5) | 21.45 (17.75-25.5) | 27.32 (23-33) | <0.01 |
| SAPS II | 59.85 (50.5-68.5) | 56.23 (49.5-59) | 64.05 (58-74) | 0.03 |
| SOFA | 9.54 (7-12) | 8.5 (6.75-10.25) | 10.74 (7-13) | 0.04 |
| Biomarkers (IQR) | ||||
| Ferritin (NG/mL) | 895.33 (376.5-1259) | 724.91 (349.25-901.25) | 1103.61 (685.75-1329) | 0.07 |
| CRP (MG/L) | 179.31 (73.1-256.5) | 170.81 (60.78-255.8) | 190.32 (103.95-258.55) | 0.59 |
| D-dimer (MG/L FEU) | 21.93 (2.12-25.65) | 13.71 (1.66-19.03) | 31.45 (4.11-56.27) | 0.05 |
| Procalcitonin (NG/mL) | 6.35 (0.473-4.34) | 6.43 (0.19-2.37) | 6.27 (0.825-7.205) | 0.97 |
| Lactate (mmol/ L) | 1.25 (0.905-1.51) | 1.25 (0.87-1.56) | 1.26 (0.92-1.39) | 0.94 |
| Fibrinogen (MG/DL) | 556.53 (411.5-685.75) | 555.9 (387-708) | 557.2 (429.5-685.75) | 0.98 |
| Respiratory variables (IQR) | ||||
| P/F Ratio (ml/cmH2O) | 159.15 (98-202.5) | 187.5 (125.75-224) | 126.4 (81-156) | <0.01 |
| Static Compliance (Torr) | 41.05 (28.38-45.1) | 42.19 (29.88-45.05) | 39.72 (28-45.27) | 0.70 |
| Treatment- N (%) | ||||
| ECMO | 2 (4.9%) | 1 (4.5%) | 1 (5.3%) | 0.91 |
| Antibiotics | 41 (100%) | 22 (100%) | 19 (100%) | NA |
| Hydroxychloroquine | 38 (92.7%) | 21(95.5%) | 17(89.5%) | 0.47 |
| Antiviral | 1 (2.4%) | 1 (4.5%) | 0 (0%) | 0.35 |
| IL6- Antagoinist | 2 (4.9%) | 0 (0%) | 2 (10.5%) | 0.12 |
| Convalescent Plasma | 16 (39.1%) | 7 (31.8%) | 9(47.4%) | 0.31 |
| Steroids | 28 (68.3%) | 17 (77.3%) | 11 (57.9%) | 0.18 |
| Outcome Mean (IQR) | ||||
| 28-day vent free day | 8.71 (0-19) | 16.23 (10.5-22.25) | NA | <0.0001 |
| 30-day hospital free day | 4.15 (0-8.5) | 7.32 (0-12.5) | NA | <0.001 |
Results:
A total of 44 cytokines/chemokines were measured, and 13 were associated with the selected outcomes (Figure 1-A); complete dataset including covariables, outcomes and cytokines expressions can be found in https://figshare.com/s/5602c77cd1b7559b1593. After adjustment for multiple comparisons/false discovery rate (FDR), only one entity, Chemokine (C-C motif) ligand 19 (CCL19) also known as macrophage inflammatory protein-3-beta (MIP-3-β), remained significantly associated with worse outcomes (Figure 1-B and C). Higher CCL19 levels were also positively correlated with APACHE II (p<0.001, r20.19), SOFA (p<0.001, r2 0.3) and SAPS II (p=0.0012, r20.24) severity scores, and with patients’ hypoxemia as reflected by the P/F ratio (p=0.0025, r20.21), suggesting that it was a measure of COVID19 disease burden.
Figure.
A: Bar graph representing the cytokines/chemokines associated with the main outcome measure, and the directionality of the regulation (red, increased in the worse outcome group, blue, decreased in the worse outcome group). These associations were found to have a p value of <0.05 before correcting for multiple comparisons/false discovery rate. Analysis was done with ANOVA test. B: After adjusting for multiple comparisons and false discovery rate, only CCL19 remained significantly associated with the main outcome. C: Comparison of CCL19 detected quantities between group 1 (worse outcome) versus group 2 (better outcome). ** p=0.009
Discussion:
Here, we describe a complex cytokine/chemokine landscape associated with COVID19-ARDS and show that higher CCL19 plasma expression is significantly associated with worse outcomes. Although to our knowledge CCL19 plasma expression has not yet been reported in the context of COVID19 infection, higher expression was previously found in a ferret model of SARS-CoV infection5; and also associated with human immunodeficiency virus (HIV) infection progression and viral load6. Importantly, while CCL19 regulates the formation of bronchus-associated lymphoid tissue (BALT)7, its pulmonary expression was shown to be necessary to support local immunity in response to influenza virus infection8. Although our finding requires corroboration, we believe circulating levels of CCL19 from patients with severe COVID19 infection could have important implications, both as a biomarker of poor prognosis, and as a potential therapeutic target for immune modulation strategies focused on the COVID19 patients developing respiratory failure. Moreover, other cytokines found associated with the main outcome could, if confirmed, provide important insight into the mechanism regulating COVID19-associated respiratory failure and mortality.
Conclusion:
Elevated CCL19 levels predict worse outcomes in a prospective cohort of COVID19 patients suffering from ARDS and on mechanical ventilation. This finding requires corroboration and could lead to the identification of an early biomarker of worse outcome, and a potential target to influence disease course.
Acknowledgments
Funding: Part of the results reported herein have been funded by NHLBI of the National Institutes of Health under the award number K01-HL130704 (AJ), and by the Collins Family Foundation Endowment (AJ); NIH/NHLBI 5R01HL049426 (HAS); NIH/National Institute of General Medical Sciences Grant 1R01GM124133 (APA); P41 GM108538 (KAO, ES, JJC).
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