key: cord-1011183-yl8s6hvp authors: Yang, Boyi; Chang, Xiaoyan; Huang, Jiabao; Pan, Wen; Si, Zhilong; Zhang, Cuntai; Li, Hong title: The role of IL-6/lymphocyte ratio in the peripheral blood of severe patients with COVID-19 date: 2021-03-12 journal: Int Immunopharmacol DOI: 10.1016/j.intimp.2021.107569 sha: 63fa8739e70c414711fb90967e1770f538ad0901 doc_id: 1011183 cord_uid: yl8s6hvp Background To investigate the prognostic value of a novel immune-inflammatory index, the interleukin-6-to-lymphocyte ratio (IL-6/LY), with the clinical outcomes of severe coronavirus disease 2019 (COVID-19) cases Methods A cohort study of COVID-19 patients in Tongji Hospital, from January 2020 to February 2020, was evaluated. Kaplan–Meier method and the log-rank test was performed to analyze survival data. Univariate and multivariate analyses were performed with COX proportional hazard regression model. The primary and secondary outcomes were in-hospital mortality and multiple organ dysfunction syndrome (MODS), respectively Results Total 320 adult patients were enrolled in our analyses. Patients were divided into low IL-6/LY group and high IL-6/LY group based on the cutoff value with 2.50. The Kaplan-Meier survival curves showed that high-value group (IL-6/LY≥2.50) had a greater risk of poor prognosis (P<0.001, respectively). Multivariate analysis indicated that IL-6/LY was the independent risk predictor for in-hospital mortality (hazard ratio [HR]=3.404, 95% confidence interval [CI]=1.090-10.633, P=0.035) and MODS development (HR, 4.143; 95%CI, 1.321-12.986, P=0.015). Meanwhile, IL-6/LY was positively correlated with the MuLBSTA score (r=0.137, P=0.031), suggesting that IL-6/LY was associated with long-term mortality (90-day). Furthermore, kinetic analysis revealed that the dynamic changes of inflammatory immune indexes were related to the severity of the disease Conclusions The elevated IL-6/LY was related with the increased risk of poor prognosis. Not only that, IL-6/LY could be used for risk stratification and early clinical identification of high-risk patients In December 2019, the outbreak of coronavirus disease 2019 (COVID- 19) , caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly throughout the world [1] . Considering the present epidemic situation, the World Health Organisation (WHO) declared a global pandemic on March 11, 2020 [2] . The raging infectious disease has caused a serious threat to human health. This brings the global cumulative numbers to 110.7 million cases and over 2.4 million deaths since the start of the pandemic [3] . SARS-CoV-2 is a beta-coronavirus, similar to other two known virus: severe acute respiratory syndrome-CoV (SARS-CoV) and Middle East Respiratory Syndrome-CoV (MERS-CoV), which have caused potentially fatal infections over the last 20 years [4] . The whole genome sequencing indicated that SARS-CoV-2 was very closely related to SARS-CoV [5] . Although SARS-CoV-2 is less lethal than MERS-CoV and SARS-CoV, it has stronger interpersonal transmission ability [6, 7] . Most patients with COVID-19 were asymptomatic or presented mild to moderate symptoms, however, about 10-20% of cases developed severe symptoms, characterized by the rapid development of acute respiratory distress syndrome (ARDS), sepsis and/or multiple organ failure [2] . In particular, the elderly and those with comorbidities tended to develop severe symptoms [8] , which might be due to their weaker immune function. Till now, the pathogenicity of COVID-19 has not been completely understood, and the underlying mechanism leading from mild to severe cases remains unclear. Recently, it has been speculated that the cytokine storm and immune dysfunction was closely related to the rapid disease progression [9] . Researchers found that the levels of infection-related biomarkers played vital roles in severe cases of COVID-19 [10] . Moreover, previous studies showed that lower lymphocyte counts, especially decreased levels of T lymphocyte subsets were linked to severe cases, accompanied with elevated levels of pro-inflammatory cytokines like interleukin-6 (IL-6) [11, 12] These suggested that immune dysfunction and cytokine dysregulation might be the key factors in the progression of the disease. In the current situation, the identification of disease progression of COVID-19 mainly depends on the clinical manifestation, while effective biomarkers have not been proposed. It was important to find sensitive biomarkers to identify critically ill patients in a timely and effective manner. As mentioned above, elevated IL-6 levels and lymphopenia were correlated with severity of disease [11, 12] . The IL-6-to-lymphocyte ratio (IL-6/LY) based on the two factors may reflect the imbalance of inflammation response and immune dysfunction in the body more comprehensively. In our study, we investigated the predictive effect of the new immune-inflammatory complex index on the prognosis of COVID-19, in order to provide positive help for clinical risk assessment. A total of 320 adult patients with severe COVID-19 pneumonia who were hospitalized in Sino-French New City Branch of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from January 2020 to February 2020, were enrolled in our retrospective study. Diagnosis of confirmed 5 COVID-19 was based on the interim guidance issued by WHO [13] . The definitions of severe illness associated with COVID-19 were as follow: fever or suspected respiratory infection; plus one of the following: 1) respiratory rate > 30 breaths/min; 2) severe respiratory distress; 3) or SPO 2 ≤ 93% on room air. Moreover, patients with suspected COVID-19, patients whose age was below 18 years old, or the patients died within 24 hours of admission were excluded. The ethics Committee of Tongji Hospital approved this study (TJ-IRB20200362) and waived the requirement for written informed consent due to the massive contagion outbreak of this infectious disease. We reviewed the electronic medical records system in hospital and collected the clinical information of all participants, including age, sex, smoking history, past medical histories (such as coronary artery disease, hypertension and diabetes). In addition, the data of initial common symptoms were gathered at the time of admission. The treatments during hospitalization (like oxygen therapy, pharmacotherapy and invasive therapies) were included in our data as well. What's more, the complications were destined to record. Venous blood samples were routinely collected from all patients during the time of hospitalization. The data were checked by two trained physicians independently. Laboratory findings included complete blood counts, coagulation function (prothrombin time, activated partial thromboplastin time, D-dimer), cardiac function (high sensitivity cardiac troponin I, N-terminal pro brain natriuretic peptide), liver 6 function (aminopherase, creatine kinase, lactate dehydrogenase), kidney function (urea nitrogen, creatinine), lipid profile and immuno-inflammatory indices. The primary endpoint of our study was in-hospital mortality, which was identified as the all-cause death happened during hospitalization. The secondary end point was incidence of multiple organ dysfunction syndrome (MODS), which was identified as acute dysfunction or failure happened in more than one system simultaneously or sequentially because of the severe diseases (such as serious infection). Clinically, MODS was common in lung, kidney, liver, heart, central nervous system, immune system and hematologic system [14] . The IL-6/LY was calculated as follow: IL-6/LY = IL-6 / lymphocyte counts. The neutrophil to lymphocyte ratio (NLR) was calculated as follow: N/L = neutrophil counts / lymphocyte counts. Continuous variables were described as mean ± standard error and tested for normal distribution by the Kolmogorov-Smirnov test. Using independent samples t test or Mann-Whitney U-test to compare the continuous variables between groups. Categorical variables were described as frequency rates and percentages and compared with the chi-square test. The correlation between IL-6/LY and MuLBSTA score was tested by using Spearman correlation coefficient. The optimal cut-off point for IL-6/LY was calculated by receiver operating characteristic (ROC) curve. Kaplan-Meier method and the log-rank test was performed to analyze survival data. Univariate and multivariate analyses were conducted with COX proportional hazard regression model. Any variables examined in the univariate 7 analysis for which the P value was <0.10 or several established risk factors were contained in the multivariate model. A P value <0.05 was considered statistically significant. All data were analyzed using SPSS version 22.0 and Graphpad 6.0 software. Totally, 320 severe patients with COVID-19 were enrolled in our study and they all met the diagnostic criteria of severe COVID-19 patients as described above. In accordance with the cut-off value of IL-6/LY, the whole population was divided into low IL-6/LY group (207 patients, 100 males) and high IL-6/LY group (113 patients, 40 males). The demographics and clinical characteristics of study population were shown in Table 1 . The patients in high IL-6/LY group were older, smoking more and with more comorbidities of coronary heart disease, diabetes and chronic lung disease (P<0.050, respectively). Additionally, more patients in the high IL-6/LY group experienced moderate to high-grade fever (P<0.050), with longer days from onset to hospitalization (P=0.023), more complications (P<0.001), and higher occupancy ratio of oxygen therapy and invasive treatment applications (P<0.050, respectively). Conversely, patients in the low IL-6/LY group stayed longer in hospital (P<0.001). The main clinical manifestations were dyspnea and mild-grade fever, and more patients in low IL-6/LY group received pharmacotherapy in hospital (P<0.050). The laboratory characteristics of the patients were summarized in Table 2 . Higher levels of white blood cells, neutrophil counts, monocyte counts, prothrombin time, D-dimer, hypersensitive cardiac troponin I, NT-proBNP, ALT, AST, globulin, 8 triglyceride, creatine kinase, LDH, urea, creatinine, high-sensitivity C-reactive protein (Hs-CRP), ESR, ferritin, procalcitonin, IgA, IgG, IL-2R, IL-6, IL-8, IL-10, TNF-α and MulBSTA score were found in high IL-6/LY group patients (P<0.050, respectively), comparing to those in low IL-6/LY group. However, the value of lymphocyte, platelet, albumin, total cholesterol, HDL-C, LDL-C, C3 and C4 was lower in high IL-6/LY group compared with that in low IL-6/LY group (P<0.050, respectively). The following categorical variables were entered in a forward stepwise COX had the best predictive ability ( Table 4) . The Kaplan-Meier survival curves for high-value and low-value groups were shown in Figure 2 . It was suggested that the high-value group (IL-6/LY≥2.50) has a greater risk in poor prognosis (P<0.001, respectively). Interestingly, we also found that IL-6/LY was positively correlated with the MuLBSTA score which was used to predict the mortality risk of viral pneumonia ( r=0.137, P= 0.031; Figure 3 ). Among our population, there were 105 intensive care patients and 215 non-intensive care patients. As the Figure 4 A-B showed, the blood LY% and LY were much lower in intensive care unit (ICU) group than those in non-ICU group on admission, and the difference was statistically significant (P<0.001, respectively). Besides, the LY% and LY gradually declined during the first three days of hospitalization in non-ICU group, while since then, they increasingly rose to normal levels upon discharge (P<0.001, respectively). In contrast, the above trend did not appear in ICU patients and both the LY% and LY still remained at a low level until discharge. In addition, significantly increased in IL-6 level and IL-6/LY were observed in ICU group compared with those in non-ICU group at several time points except for the first three days in hospital (the day of at admission, four to seven days after admission, eight to eleven days after admission, twelve to fifteen days after admission, 27 before discharge, all P<0.001). The difference between ICU group and non-ICU group was significant at the time point of 4-7 days and became even greater before discharge (Figure 4 C-D) . In current study, we found that patients with elevated IL-6/LY (≥2.50) was the independent risk factor for in-hospital mortality and the development of MODS in severe COVID-19 patients. Besides, our results showed that the age along with LY% were also the predictors of in-hospital mortality and the diabetes, prothrombin time, and NT-ProBNP were significant associated with higher likelihood of MODS development. Meanwhile, the dynamic changes of the inflammatory biomarkers could reflect the clinical severity in patients with COVID-19. A series of evidences showed that the uncontrolled inflammatory response and immunity dysregulation were the prominent feature of critical ill COVID-19 patients [2, 15] . In addition, several other researches also proved that some seriously ill COVID-19 patients have increased cytokine profile similar to cytokine storm in severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) [16] [17] [18] . Cytokine storm stems from the coronavirus infected place and then spread throughout the organism by mass of inflammatory cells infiltration which in turn lead to acute lung injury, ARDS and death [17, 18] . It was reported that higher levels of inflammatory parameters, especially IL-6 could be shown in severe COVID-19 patients, which was consistent with previous studies [8, 19, 20] . IL-6 has multiple effects in regulating inflammation. Cifaldi et al. has reported that elevated IL-6 level was linked to impaired cytolytic function by overstimulating the immune system and finally might result in multiple organ failure [21] . 28 Furthermore, significantly reduced of T cells counts have been found in severe COVID-19 patients in recent studies [22, 23] . Researchers identified that very few lymphocytes were infiltrated in the alveolar of damaged lung tissue [10, 24, 25] . A similar decrease in lymphocyte counts and the subset of T cells could be seen in SARS patients according to previous investigations [26, 27] . However, the exact mechanism of lymphopenia in severe COVID-19 patients remains still unclear. Previous body biopsies reported that the COVID-19 patients' secondary lymphoid tissues had been destroyed [10, 24, 25] . Zhang et al. speculated that lymphocytes were directly invaded by virus infection or indirectly damaged by cytokine storm which induced by immune response [24] . And a substantial decrease in lymphocytes revealed that the immune cells may be consumed by the viruses and the body's cellular immune function may be restrained [28, 29] . It suggested that COVID-19 infection can lead to immune dysfunction through affecting the subsets of T cells [30] . inflammatory monocytes (CD14 + CD16 + with high expression of IL-6) existed particularly in ICU patients [31] . Therefore, lots of these pathogenic T cells and inflammatory monocytes may get into the pulmonary circulation and arouse inflammatory storm which probably prevents alveolar gas exchange and contributing to the high mortality of severe COVID-19 patients [32] . Given their weight during the course of COVID-19, our study developed a novel biomarker, named as IL-6/LY, in order to estimate condition, evaluate prognosis and conduct risk stratification. As we know, this was the first research for exploring the effect of IL-6/LY on predicting the clinical outcomes for COVID-19 cases. According to existing research results, we proposed potential mechanisms of high IL-6/LY was resulted from the increased IL-6 and the decreased lymphocyte counts. Recent studies' results would give some explanations. Wan S et al found that patients with severe COVID-19 patients were more likely to have higher IL-6 levels than those mild COVID-19 cases [23] . It was also showed that lymphopenia was one of the important features of COVID-19 infection and which was also the common ground in most severe patients [1] . As shown in another study, IL-6 could suppress the T cell activation, which may explain the decrease of lymphocyte [33] . Furthermore, The dynamic changes of IL-6/LY in patients revealed that the magnitude of the immune response dysregulation was related to the severity of COVID-19 patients. Herby, the physicians could identify the specific subpopulations of COVID-19 patients who were at greater risk for unfavorable outcomes at an early stage. What's more, in our study, the relationship between other immunoinflammatory parameters and poor prognosis were also evaluated. We found that individual variable (such as WBC, Hs-CRP, procalcitonin, ferritin and interleukins) had an influence on the occurrence of clinical outcomes partly. However, after adjustment for potential confounders, the results statistically supported the conclusions that the incidence of high IL-6/LY was significantly correlated with the poor prognosis of the disease. Not only that, older age and comorbidities were proved to be in connection with severe COVID-19 [35] . Weina Guo et al suggested that COVID-19 patients with diabetes had a poor prognosis and diabetes was proposed as a risk factor for the progression of COVID-19 cases [36, 37] . Clinically, infected by SARS-CoV-2 caused multiple system organ failure, such as the hematologic system [38] [39] [40] . Previous studies showed that prothrombin time was positively correlated with 28-day mortality [41] . Besides, a retrospective study showed that NT-proBNP was an independent risk factor for in-hospital death in patients with severe COVID-19 [42] . In fact, these results were in consistent with our findings. We have to acknowledge that there were some limitations in this study. Firstly, the retrospective design of the study set a limit to the convincement of our study. Due to the nature of our study, the results must be explained with caution, given the possibility of confounders. Secondly, because of the objective conditions, the patients were not followed up outside of hospital. And we did not study the relationship between IL-6/LY with the long-term outcomes. Thirdly, our sample size was small. Therefore, prospective clinical studies with larger population were needed. 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The authors declared that there is no conflict of interest. All authors read and approved the final manuscript.