key: cord-0977327-j5csecuu
authors: Wang, Guo-Lin; Gao, Hui-Xia; Wang, Yu-Ling; Wei, Xiao; Liu, Yu-Zhen; Lu, Jian-Hua; Li, Li; Wang, Hai-Bin; Zhao, Lei; Rong, Yan-Xiao; Yao, Lin; Duan, Li-Juan; Anderson, Benjamin D.; Ma, Mai-Juan; Dai, Er-Hei; Zhao, Xiang-Na
title: Serum IP-10 and IL-7 levels are associated with disease severity of coronavirus disease 2019
date: 2021-03-19
journal: Cytokine
DOI: 10.1016/j.cyto.2021.155500
sha: b7dff236acf1cd682f233a663acd435e347bb266
doc_id: 977327
cord_uid: j5csecuu

We quantified the serum levels of 34 cytokines/chemokines in 30 patients with SARS-CoV-2 infection. Elevated levels of IP-10 and IL-7 were detected in the acute and convalescent stages of the infection and were highly associated with disease severity.

The coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a severe threat to the international community.

Patients with COVID-19 are clinically characterized by fever, cough, fatigue, sputum production, lymphocytopenia, and radiographic evidence of pneumonia [1] . Death may result from sepsis, respiratory failure, and acute respiratory distress syndrome (ARDS) due to alveolar damage or multiple organ dysfunction caused by systemic inflammation [1] Cytokines/chemokines have long been thought to play an essential role in immunity and immunopathology during virus infections. Although increased serum levels of several inflammatory cytokines and chemokines were associated with disease severity and death of SARS-CoV-2 [1] [2] [3] [4] [5] , the results of these studies were discrepancies. The pathogenesis of COVID-19 is poorly understood, and factors associated with severity remain elusive. Therefore, to further understand the immuno-pathogenesis of SARS-CoV-2, we studied the expression profile of cytokines/chemokines in the serum of patients with COVID-19.

From January to February 2020, a total of 30 COVID-19 patients and individuals with asymptomatic infection of SARS-CoV-2 were identified at the Fifth Hospital of Shijiazhuang, Shijiazhuang City, Hebei Province, China. All COVID-19 patients and individuals with asymptomatic infection of SARS-CoV-2 were laboratory-confirmed by real-time reversetranscription polymerase chain reaction (RT-PCR) using a SARS-CoV-2 nucleic acid detection kit (Cat No. DA0930-DA0932, DAAN GENE Ltd., Guangzhou, China). After the confirmation diagnose, COVID-19 patients and individual with asymptomatic infection of SARS-CoV-2 were recruited to participate the study. After obtaining informed consent, serum samples were collected from participants at the acute and convalescent stages of infection.

We defined the definition of acute and convalescent stages based on the symptoms of the patients after infection. The acute stage always correlated with fever, cough, fatigue, expectoration, dyspnea, chills, and so on, and the convalescent stages correlated symptoms were significantly improved. Therefore, the acute stage was defined as within 2 weeks after symptom onset for symptomatic patients and after first RT-PCR positive RNA detection of SARS-CoV-2 for asymptomatic infection, and the convalescent stage was defined between 2 and 4 weeks after symptom onset for symptomatic patients and after first RT-PCR positive RNA detection of SARS-CoV-2 for asymptomatic infection. The disease severity were categorized depending on the severity of illness according to the Diagnosis and Treatment Plan for New Coronavirus Infected Pneumonia (Trial Seventh Edition) by the National Health and Health Commission of China [6] . Twenty-four serum samples from healthy adults with matched age and sex were used as normal control samples. The exclusion criteria for serum samples from healthy adults were as follows: individual who had an acute or chronic infectious disease, any clinically significant disorder (e.g. depressive disorder and anxiety disorder), any medication with known influence on immunological factors. The study was conducted following the Declaration of Helsinki, and the Ethical Committee of the Fifth Hospital of Shijiazhuang approved this study (2020008).

The concentration of serum cytokines/chemokines (interferon alpha [IFN-α], IFN-γ, IL 

Comparison of clinical parameters among severe, moderate, and asymptomatic patients was calculated by non-parametric Mann-Whitney test (for continuous variables) or Pearson χ 2 test on cross table (for categorical variables). For multiple groups, comparisons were made by two-way ANOVA or non-parametric Kruskal-Wallis test followed by multiple comparisons. P < 0.05 was considered to indicate a significant difference.

Twenty-one patients (5 severe and 16 moderate) and 9 subjects with asymptomatic infection were enrolled (Supplemental Table1). Older age was associated with severe disease (Supplemental Table 1 ; P < 0.05). In addition, the decrease in lymphocytes was observed in patients with severe disease (Supplemental Table 2 

Among 34 cytokines/chemokines tested, the levels of 23 cytokines/chemokines were undetectable in the serum of patients and controls. Therefore, the remaining 11 cytokines/chemokines (IP-10, Eotaxin, RANTES, MCP-1, SDF-1α, MIP-1β, MIP-1α, Gro-α, IL-7, IL-8, and IL-18) were further analyzed. We first analyzed expression profiles of the 11 cytokines/chemokines between acute and convalescent serum ( Figure 1A ). The levels of IP-10 and Eotaxin were significantly higher in both acute (P=0.0001 and 0.030, respectively) and convalescent (P = 0.008 and 0.008, respectively) sera compared with control subjects. The level of IL-7 was significantly higher in the acute serum compared to the convalescent serum (P = 0.025) and control subjects (P = 0.002). In contrast, there were significantly decreased levels of GRO-α in the acute (P = 0.0317) and convalescent (P = 0.0143) sera and RANTES in the convalescent (P = 0.0036) sera compared with the control subjects. No significant differences were observed for the levels of the other six cytokines/chemokines between patients and control subjects (Supplemental Figure 1) .

We further sub-analyzed the levels of these 11 cytokines/chemokines between the acute and convalescent phases of each group of patients ( Figure 1B) . For severe patients, the levels of IP-10 and IL-7 were significantly higher in the acute and convalescent sera than control subjects. Besides, the level of IP-10 in the acute serum was substantially higher than the convalescent serum (P = 0.029). RANTES level in acute and convalescent sera was lower compared with controls (P = 0.038 and 0.017, respectively). A higher level of Eotaxin was observed in the convalescent serum compared with controls (P = 0.02), but no difference was found compared with the acute serum. For moderate patients, a significantly higher level of IP-10 was also detected in the acute serum compared with the convalescent serum and control subjects (P = 0.042 and 0.004, respectively). We also found a higher level of MCP-1 in the acute serum and of Eotaxin in the convalescent serum compared with control subjects (P = 0.002 and 0.013, respectively). For patients with asymptomatic infection, IL-7 level in the acute serum was significantly higher than compared to the control subjects (P = 0.026), whereas patients at the acute phase had lower levels of SDF-1α and MIP-1α compared with controls (P = 0.028 and 0.034, respectively).

Next, we evaluated the levels of these 11 cytokines/chemokines for disease severity in acute and convalescent sera. In the acute phase ( Figure 1C) , severe patients had higher levels of IP-10 and IL-7 compared with moderate (P = 0.014 and 0.002, respectively) and asymptomatic (P < 0.0001 and = 0.021, respectively) patients as well as control subjects (P < 0.0001 and = 0.0002, respectively). We observed a higher level of MCP-1 in moderate patients compared with other groups. At the convalescent phase, severe patients had higher levels of IP-10 and IL-7 compared with moderate (P = 0.0005 and 0.0003, respectively) and asymptomatic patients (P = 0.020 for IL-7) and controls (P < 0.0001 and = 0.0005, respectively).

Because several of the 23 undetectable cytokines were detected in three patients, we tried to describe the dynamic levels of these cytokines in sequential serum samples from these three patients. As shown in Figure We also found a quick decline level for IL-6 compared with other cytokines. In contrast, only three cytokines (IL-1α, IL-15, and IL-22) were elevated in serum within ten days after the first SARS-CoV-2 detection of patient C, who was asymptomatic. It seems that the levels of IL-1α and IL-22 had an increasing trend but a declining pattern for IL-15.

In this study, we measured the cytokine/chemokine responses in serum of patients with SARS-CoV-2 infection. Our results revealed a minimal pro-inflammatory cytokine/chemokine response after infection of SARS-CoV-2 as there were no increase in levels of most proinflammatory cytokines/chemokines that have been implicated in immune reactions against viruses, such as IFN-γ, TNF-α, IL-1β, IL-6, and so on.

Previous studies have shown that increased amounts of pro-inflammatory cytokines in serum (e.g., IL-1β, IL-6, IL-12, IFN-γ, IP-10, and MCP-1) were associated with disease severity in SARS patients [7] [8] [9] . MERS-CoV infection could also induce increased concentrations of pro-inflammatory cytokines/chemokines (IFN-γ, TNF-α, IL-6, IL-15, IL-17, IL-1RA, IP-10, and MCP-1) [10, 11] . For the patients with SARS-CoV-2 infection, high amounts of IL-1β, IFN-γ, IP-10, and MCP-1 were observed, and high concentrations of GCSF, IP-10, MCP-1, MIP-1A, and TNF-α were associated with disease severity (i.e., higher levels in intensive care unit (ICU) patients) [1] [2] [3] [4] [5] . However, most pro-inflammatory cytokines/chemokines, such as IL-2, IL-6, IL-10, IFN-γ and TNF-α [3, [12] [13] [14] , were not detected in patients of our study. A similar result was also observed in another study where only minimal pro-inflammatory cytokines/chemokines were found in a COVID-19 patient [15] .

In the present study, although 11 (IP-10, Eotaxin, RANTES, MCP-1, SDF-1α, MIP-1β, MIP-1α, Gro-α, IL-7, IL-8, and IL-18) cytokines/chemokines were detected in patients, only 5 of them had a showed a significant elevated or decreased level compared with control subjects, including increased levels of IP-10, Eotaxin, and IL-7, and declined levels of RANTES and GRO-α. The Increased plasma levels of IL-7 and IP-10 were also observed in intensive care unit (ICU) patients compared with non-ICU patients [16] . Recent studies also reported that higher level of IL-7 was associated with the severity of COVID-19 [12, 17, 18] . In this study, severe patients showed a decreased of lymphocytes and an elevated level of IL-7, indicating that IL-7 was associated with lymphocytes returning to a reference level, appearing to reverse a pathologic hallmark of COVID-19. IP-10 has been reported as an excellent biomarker for the prediction of COVID-19 progression and can be related to the risk of death in COVID-19 patients [5, 14, 19, 20] . Consistent with the previous study, we found that IP-10 was augmented markedly in the serum of patients at an early stage but remained at a high level during the convalescent stage in severe and moderate patients. In addition, a similar profile of IL-7 was observed. IL-6 was recognized as the main cytokines related to the severity of COVID-19 [12, [21] [22] [23] , but we did not detected IL-6 in both patients and control subjects in this study, the possible reason is the different methods of measurement of the cytokines.

The limited number of patients recruited in this study, however, was an obstacle to reaching a conclusion. The second weakness of this study is the absence of viral load data. 

Pathological findings of COVID-19 associated with acute respiratory distress syndrome, The Lancet

Clinical features of patients infected with 2019 novel coronavirus in

Clinical and immunological features of severe and moderate coronavirus disease

Dysregulation of immune response in patients with COVID-19 in Wuhan, China

Plasma IP-10 and MCP-3 levels are highly associated with disease severity and predict the progression of COVID-19

Diagnosis and Treatment Plan for New Coronavirus Infected Pneumonia

Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome

Characterization of cytokine/chemokine profiles of severe acute respiratory syndrome

Temporal changes in cytokine/chemokine profiles and pulmonary involvement in severe acute respiratory syndrome

MERS-CoV infection in humans is associated with a pro-inflammatory Th1 and Th17 cytokine profile

Immune Responses to Middle East Respiratory Syndrome Coronavirus During the Acute and Convalescent Phases of Human Infection

Serum Cytokine and Chemokine Profile in Relation to the Severity of Coronavirus Disease

Should we stimulate or suppress immune responses in COVID-19? Cytokine and anticytokine interventions

Interferon-γ-induced protein 10 (IP-10) and serum amyloid A (SAA) are excellent biomarkers for the prediction of COVID-19 progression and severity

Breadth of concomitant immune responses prior to patient recovery: a case report of non-severe COVID-19

Clinical features of patients infected with 2019 novel coronavirus in

Association of Interleukin 7 Immunotherapy With Lymphocyte Counts Among Patients With Severe Coronavirus Disease

Immune monitoring of interleukin-7 compassionate use in a critically ill COVID-19 patient

IP-10 and MCP-1 as biomarkers associated with disease severity of COVID-19

Observational cohort study of IP-10's potential as a biomarker to aid in inflammation regulation within a clinical decision support protocol for patients with severe COVID-19

IL-6 and IL-10 as predictors of disease severity in COVID-19 patients: results from meta-analysis and regression

Laboratory findings in SARS-CoV-2 infections: State of the art

Dynamic Interleukin-6 Level Changes as a Prognostic Indicator in Patients With COVID-19

Ma drafted the manuscript. Benjamin D. Anderson reviewed and edited the manuscript. All authors reviewed and approved the final manuscript

We thank all patients for their participation in the study. This study was supported by the 

The authors declared no conflicts of interest.

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: