key: cord-0743846-ooalz8gc
authors: Rubina, K. A.; Shmakova, A. A.; Shabanov, A. K.; Andreev, Y. V.; Borovkova, N. V.; Kulabukhov, V. V.; Evseev, A. K.; Popugaev, K. A.; Petrikov, S. S.; Semina, E. V.
title: Novel prognostic determinants of COVID-19-related mortality: a pilot study on severely-ill patients in Russia
date: 2021-04-07
journal: nan
DOI: 10.1101/2021.04.01.21254688
sha: 1788475199f92d1b10e8145705e39f15925ed4f8
doc_id: 743846
cord_uid: ooalz8gc

COVID-19 pandemic has posed a severe healthcare challenge calling for an integrated approach in determining the clues for early non-invasive diagnostics of the potentially severe cases and efficient patient stratification. Here we analyze the clinical, laboratory and CT scan characteristics associated with high risk of COVID-19-related death outcome in the cohort of severely-ill patients in Russia. The data obtained reveal that elevated dead lymphocyte counts, decreased early apoptotic lymphocytes, decreased CD14+/HLA-Dr+ monocytes, increased expression of JNK in PBMCs, elevated IL-17 and decreased PAI-1 serum levels are associated with a high risk of COVID-19-related mortality thus suggesting them to be new prognostic factors. This set of determinants could be used as early predictors of potentially severe course of COVID-19 for trials of prevention or timely treatment.

failure and even death 2-4 . Older age, diabetes and cardiovascular comorbidities are among the 44 highest risk factors influencing the disease severity and the clinical outcome of SARS-CoV-2 45 infection 4,5 . However, severe COVID-19 has been registered not only in elderly patients or 46 patients suffering from comorbidities, but also in apparently healthy young people 6,7 . The 47 development of new prognostic algorithms for differential diagnostics to predict and reduce the 48 severity and complications of COVID-19 remains essential. 49 The data obtained in the present study reveal novel prognostic factors associated with high 50 risk of COVID-19-related death overtime in patients with severe COVID-19. These include: the 51 elevated dead lymphocyte counts, decreased early apoptosis of lymphocytes, decreased CD14+, 52

HLA-Dr+ (human leukocyte antigen D related) monocytes, increased JNK (c-Jun N-terminal 53 kinase) expression in PBMCs (peripheral blood mononuclear cell), increased IL-17 and decreased 54 PAI-1 (plasminogen activator inhibitor-1) serum level. The prognostic value of each determinant 55

can potentially impact decision-making on the level of healthcare and require more extensive 56

follow-up strategies. 57

To the best our knowledge this is the first integrated study based on the Russian population 58 aimed to reveal the original prognostic COVID-19-related death factors. 59

The present study was a single-center retrospective study carried out on a cohort of 52 62 symptomatic adult patients with the confirmed SARS-CoV-2 infection, admitted to intensive care 63 unit (ICU) of N.V. Sklifosovsky Research Institute for Emergency Medicine, Moscow from April 64

to July 2020. Demographic, clinical, and laboratory data were recorded at admission. The study 65 was approved by the ethics committee of Sklifosovsky Research Institute of Emergency Medicine 66 of the Moscow Healthcare Department (protocol #5-120, issued on 01.04.2020). The need for 67 written patient consent was waived by the ethics committee of the hospital because laboratory 68

investigations were conducted according to the local standard. The local ethical committee 69 approved the retrospective study. 70 Nasopharyngeal swabs were collected from all patients, followed by RT-PCR assay to 71 confirm the SARS-CoV-2 infection. Philips Ingenuity Core 128 CT Scanner (Netherlands) was 72 used for all examinations. In all cases, semi-quantitative CT scoring (severity ranging from 0 to 4) 73 was evaluated according to the protocol recommended by Moscow Healthcare Department 74 adapted from the International Protocols and enriched with local experience 8 . 75

Routine blood tests were carried out for all patients and the following parameters were 76 evaluated: white blood cells (WBC), lymphocytes, monocytes, neutrophil, red blood cell (RBC), 77 platelet counts, levels of hemoglobin, serum total protein, albumins, globulins, bilirubin, urea, 78 creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate 79 dehydrogenase (LDH), creatine kinase (CK), prothrombin, fibrinogen, D-dimer levels, activated 80 partial thromboplastin time (APTT) and the international normalized ratio (INR). 81

In addition to blood tests routinely and periodically performed in hospital, we assessed the 83 levels of plasminogen (PLG), plasminogen activator inhibitor 1 (PAI-1), IL-1α, IL-17, 84

transforming growth factor beta (TGFβ), tumor necrosis factor alpha (TNFα), and adiponectin 85 (ADP) in blood serum. Blood serum was collected and immediately frozen. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. with CT score 4. Overall, 11 (21%) patients died.

The survival analysis was performed with time defined as a period from hospital admission 158

to the end of the follow-up (hospital discharge or death) and event defined as death or hospital 159 discharge after recovery (Figure 1) routine blood test factors with statistically significant results are presented in Figure 2 . We found, 172

that the risk of COVID-19-related death over time was significantly increased in patients with CT 173 score 3-4 at admission (P = 0.042), patients on pulmonary ventilation (P = 0.00047), patients with 174 WBC count more than 11.8•10 3 /μl (P = 0.001), neutrophil count more than 8.2•10 3 /μl (P = 175 0.00027), lymphocyte count less than 1.1•10 3 /μl (P = 0.018), increased serum levels of globulins 176 more than 30 g/l (P = 0.046), urea more than 7.2 mM (P = 0.01), CK more than 200 U/l (P = 177 0.023), prothrombin less than 78% (P = 0.023), INR more than 1.17 (P = 0.02) and D-dimer more 178 than 2 mg/l (P = 0.0078). Gender, age, BMI, tobacco smoking status and other parameters of 179 routine blood tests were not associated with statistically significant differences as revealed by 180

Kaplan-Meier analysis (Supplementary figure 2) . 181 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. ; https://doi.org/10.1101/2021.04.01.21254688 doi: medRxiv preprint

The Kaplan-Meier survival plots for the biochemical test factors being statistically 189 significant are presented in Figure 3 . We found, that the risk of COVID-19-related death over 190 time was significantly increased in patients with more than 100 dead lymphocytes in 1 µl (P = 191 0.0026), less than 5% of early apoptotic lymphocytes (P = 0.0081), less than 85% of CD14+/HLA-192 Dr+ monocytes (P = 0.029), increased expression of JNK in PBMCs (>200 MFI, P = 0.034), 193

increased serum levels of IL-17 (>7 pg/ml, P = 0.037) and decreased PAI-1 serum levels (< 40  194 pg/ml, P = 0.00073). Percentage of dead and late apoptotic lymphocytes, percentage of CD95+ 195 lymphocytes, expression of Akt, Bad, Bcl-2, Caspase-8, Caspase-9, p53 and serum levels of PLG, 196 IL-1α, TGFβ, TNFα, adiponectin were not associated with statistically significant differences in 197 survival as revealed by Kaplan-Meier analysis (Supplementary figure 3) . To identify the independent risk factors, we created multivariate Cox proportional hazards 207 survival analysis models separately for clinical characteristics and hematology results; for clinical 208 biochemical routine blood test results and for laboratory blood findings ( 1.07 (1.001-1.14), P = 0.046) and PAI-1 (HR 0.82 (0.69-0.98), P = 0.026) were found to be the 217 independent prognostic factor for COVID-19 related death. The results of univariable Cox 218 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. With the rapid spread of COVID-19 around the world since its outbreak in early 2020, it has 227 stirred up an international concern and still remains so. Chest CT imaging has been recognized to 228 play a pivotal role in monitoring COVID-19 disease progression and predicting adverse prognosis. 229 We found that CT score 3-4 at admission and pulmonary ventilation significantly increase the risk 230

of COVID-19-related death over time (Figure 2) (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. ; https://doi.org/10.1101/2021.04.01.21254688 doi: medRxiv preprint immunoglobulins 25 are statistically significant and strongly correlate with the adverse outcome in 236 COVID-19 patients. Our data also confirm the recent papers (Figure 2) Moreover, we have defined that the decrease in CD14+/HLA-Dr+ monocyte fraction are 251 significant predictors of disease severity in COVID-19 patients (Figure 3) . 252

The biological role of lymphocyte apoptosis in infectious disease is ambiguous; whether it 253 operates as an intrinsic protective mechanism or mediates a severe course of the disease still 254 remains unclear. Lymphocytes can undergo extensive and ostensibly out-of-control apoptosis 255 during severe infections, potentially contributing to immunosuppression 32 . In the majority of viral 256

infections, lymphocytes are subjected to apoptosis, which is considered to be a defense mechanism 257

preventing the spread of infection without a local inflammatory reaction 33 from COVID-19 patients as compared to control, however no association with mortality has been 264 addressed in this study 35 . Our data indicate that elevated dead lymphocyte counts are associated 265

with COVID-19 mortality (Figure 3) , however, early lymphocyte apoptosis is rather a protective 266 mechanism reflecting the intensity of the adaptive cell immune response to an infectious agent, 267

whereas a decrease in the early apoptosis of lymphocytes is an important predictor of COVID-19-268 related mortality (Figure 3) . 269 Elevated plasma D-dimer is a well-established prognostic factor for adverse outcome in 270 respiratory diseases and COVID-19 28,36,37 . Our results go along with these previously published 271 data and ascertain that the COVID-19-related death over time is significantly increased in patients 272

with augmented D-dimer (Figure 2) . Similarly, Chen and co-authors 6 reported the correlation between high concentrations of plasma 282 IL-6 and TNF-α and the severe course of COVID-19. In contrast, in the study by authors these cytokines, including IL-1β, IL-12p40, and IL-17 were undetectable in patients with 284

severe COVID-19 42 . Several clinical trials investigating interleukin inhibitors (IL-1 inhibitor 285 anakinra, and IL-6 inhibitors tocilizumab or sarilumab) revealed that IL-1 inhibition, but not IL-6 286 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In the present study IL-1α, TNF-α and TGF-β were not associated with the statistically 298 significant difference in patient survival (Supplementary figure 3) ; however, IL-17 and PAI-1 299

were determined as independent prognostic factors for COVID-19 related death (Figure 3) has been found that overproduced PAI-1 binds to specific receptors on macrophages and 315 upregulates the production of proinflammatory cytokines and chemokines, which further activate 316 the innate immune cells within the infected lungs resulting in lung damage. In turn, high hypoxic 317 environment additionally stimulates PAI-1 production creating a vicious cycle of cytokine storm 318 generated during SARS virus pathogenesis 51 . In contrast, our data indicate that COVID-19-related 319 mortality is associated with decreased PAI-1, establishing that PAI-1 being the constituent 320 component of the fibrinolytic system plays an important role in COVID-19 pathogenesis. The 321 explanation for the reduced PAI-1 content in the critically-ill patients may reside in compensation 322

for the hyperactivation of plasminogen activator system, reflecting the exhaustion of plasmin 323 inhibitors (Figure 3) .

The C-Jun NH2-terminal kinase (JNK1/JNK2) pathway activation ultimately leads to 325

various cellular effects such as inflammatory response, cell proliferation, survival or even 326 apoptosis. JNK signaling pathway was suggested to play a prominent role in immune response to 327 viral infection due to activation of several interleukins (IL-2, IL-4) and IFN-γ 55 . To uncover the 328 mechanisms of JNK-mediated inflammation in COVID-19, Shirato and Kizaki carried out an in 329

vitro study and analyzed the effects of SARS-CoV-2 spike protein S1 subunit on murine and 330 human macrophages. Exposure to S1 subunit activated pro-inflammatory mediators (nuclear 331 factor-κB (NF-κB) and JNK) signaling pathways via the activation of a toll-like receptor 4 (TLR4) 332 on macrophage surface. Pro-inflammatory cytokine (TNF-α, IL-6, IL-1β, and nitric oxide) 333 production induced by S1 was abrogated by specific inhibitors of NF-κB and JNK pathways 56 . 334

In line with the suggested strategy for inhibition of JNK pathway in COVID-19 critically-ill 335 patients as a promising therapeutic approach 57 , here we present the first clinical study revealing 336 the increased JNK expression in PBMCs to be a significant prognostic marker of COVID-19-337 related mortality (Figure 3) . It is tempting to speculate that the elevated JNK, which has been 338 demonstrated to induce TLR4-mediated apoptosis in various types of cells 56,58 , and the decreased 339 level of lymphocyte early apoptosis in severe COVID-19 patients revealed in the present study are 340

intertwined. However, this assumption needs further investigation. 341

In conclusion, this study unveiled an integrated set of immunological features and new 342 prognostic markers of patients with severe COVID-19 that are significant predictors of the related 343 mortality. The obtained results suggest a high risk of COVID-19-related death overtime in patients 344

with elevated dead lymphocyte counts, decreased early apoptosis of lymphocytes, reduced fraction 345 of CD14+/HLA-Dr+ monocytes, increased JNK expression in PBMCs, elevated IL-17 and 346

decreased PAI-1 serum levels. Gender, age, BMI, tobacco smoking status and several parameters 347 of routine blood tests (Supplementary figure 2) were not statistically significant. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. Mouse-FITC Isotype Control, A07795, Beckman Coulter) was applied as a control. 394 395 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 7, 2021. is shown in red. 401 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

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