Higher expression of monocyte chemotactic protein 1 in mild COVID-19 patients may be correlated with the inhibition of IFN signaling

Background: The level of chemokines was markedly elevated in severe COVID-19 patients. But the role of chemokines in mild COVID-19 has not yet been established. However, most of the COVID-19 patients in Shiyan City, China, had mild cases. The purpose of this study is to evaluate the level of chemokines in mild COVID-19 patients and explore the correlation between chemokines and host immune response. Methods: In this study, the level of chemokines in the serum for COVID-19 patients in Shiyan City was detected by ELISA. The expression of chemokine receptors and other signal molecules was measured by real-time PCR. Results: We rst demonstrated that COVID-19 patients are characterized by higher level of chemokines. Meanwhile, monocyte chemotactic protein 1 (MCP-1) has also shown higher expression in patients with mild cases of COVID-19. The receptor of MCP-1, CCR2, was also found to be expressed at higher level in the same mild COVID-19 patients. Finally, we found the downregulation of interferon regulatory factor 3 (IRF3) was signicantly negative correlated with the concentration of MCP-1 in mild COVID-19 patients. Conclusion: Higher expression of MCP-1 in mild COVID-19 patients may be correlated with the inhibition of IFN signaling. The nding adds our understanding to the immune-pathologic mechanisms of SARS-CoV-2 infection, and provides potential therapeutic targets and strategies. the concentration of chemokines in serum and the expression level of chemokine receptors in PBMCs from COVID-19 patients were detected, so as to evaluate the role of chemokine in mild COVID-19 in Shiyan City, China. Then we evaluated the level of IFN-β and the relationship between IRF3 and chemokine, so as to prove the correlation between chemokine and interferon signal. Our results suggested that higher expression of monocyte chemotactic protein 1 (MCP-1) in mild COVID-19 patients may be correlated with the inhibition of IFN signaling. Interferon-inducible CCR2: C-C 2; CXCR: chemokine (C-X-C motif) receptor. BMDMs: bone marrow derived macrophages.


Background
An outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread throughout the world 1, 2 . Globally, by October 2020, there have been 38,394,169 con rmed cases of COVID-19, including 1,089,047 deaths (https://covid19.who.int/). According to the epidemiological statistics, most of the COVID-19 (about 80%) are classi ed as mild 3,4 . Most of the COVID-19 patients in Shiyan city had mild cases 4 . For this reason, it is necessary to study the immunological characteristics of mild COVID-19 patients and de ne a suitable therapeutic strategy for these cases.
Immune response is the body's defense mechanism against viral infection. It involves the innate and adaptive immune responses. However, excessive immune responses after infection, also called a cytokine storm, have been found to be associated with extreme levels of pro-in ammatory cytokines. Patients infected with SARS-CoV-2 showed higher leukocyte numbers, abnormal respiratory ndings, and increased level of plasma pro-in ammatory cytokines. The direct cause of death from acute COVID-19 may be the result of a cytokine storm with damage to lungs and multiple other organs, resulting in multi organ failure 5 .
Preliminary studies have shown that SARS-CoV-2 infection may trigger a cytokine storm, and results in the increase of a variety of cytokines, including chemokines [5][6][7] . Chemokines are low-molecular-weight proteins with powerful chemo-attractant activity. They play a role in the immune cell recruitment during inflammation. Chemokines are classi ed according to their chemical structure, the C, CC CXC and CX3C families 8 . The binding of chemokines to their receptors is responsible for their chemo-attractant ability.
The chemokine receptors are seven-transmembrane-spanning, G-protein-coupled receptors. They are expressed on leukocytes and endothelial cells, etc. 9 . Serum chemokine levels were found to be elevated in patients with COVID-19, and they were even higher in those who required ICU admission, suggesting a relationship with lung damage and disease severity 10 . Although the concentration of chemokine is also elevated in mild COVID-19 patients 6 , it is not clear whether the high expression of chemokines can be used as a marker for the diagnosis and prognosis of mild COVID-19.
Type I interferons (IFNs) (including IFN-α and IFN-β) have broad-spectrum antiviral activities, which act by inducing an antiviral response and mediating adaptive immune response 11 . Infection of cells by virus induce the activation of several cellular transcription factors, such as interferon regulatory factor 3/7 (IRF3/7) and NF-κB, which activate the expression of a number of interferon-stimulated genes (ISGs) and exert antiviral effect 12,13 . The activation of transcription factors also induces the secretion of chemokines, which further recruit and coordinate speci c subsets of leukocytes, such as neutrophils and monocytes 14,15 . It means that the secretion of chemokine may be related to the release of interferon, but the speci c relationship between them in the process of SARS-CoV-2 infection has not been clearly established.
Clinically, Type I IFNs have already been approved for use in the treatment of certain cancers, autoimmune disorders, and viral infections 16 . Type I IFNs are currently in clinical trials to evaluate their ability to treat MERS-CoV 17 and therefore have been proposed for the treatment of COVID-19, but there is currently no evidence from laboratory testing against SARS-CoV-2.
In this study, the concentration of chemokines in serum and the expression level of chemokine receptors in PBMCs from COVID-19 patients were detected, so as to evaluate the role of chemokine in mild COVID-19 in Shiyan City, China. Then we evaluated the level of IFN-β and the relationship between IRF3 and chemokine, so as to prove the correlation between chemokine and interferon signal. Our results suggested that higher expression of monocyte chemotactic protein 1 (MCP-1) in mild COVID-19 patients may be correlated with the inhibition of IFN signaling.

Subjects
The severity of COVID-19 symptoms was graded according to China National Health Commission Guidelines for Diagnosis and Treatment of SARS-CoV-2 infection (seventh version) (http://www.nhc.gov.cn/yzygj/s7652m/202003/a31191442e29474b98bfed5579d5af95.shtml). 10 severe and 30 mild COVID-19 patients were recruited from Xiyuan Hospital and Renmin Hospital, Shiyan City and the study protocol received approval from the Clinical Ethics Committee of Hubei University of Medicine (2020-TH-017). Another 10 healthy subjects were recruited as a control group. All the healthy subjects were free from tumor, infection or other diseases. All individuals gave their informed consent to participate. The basic information of COVID-19 patients and healthy controls is listed in Table 1.

Isolation of Peripheral blood mononuclear cells (PBMCs)
PBMCs were isolated through density-gradient centrifugation using Ficoll-Paque (Sigma, 17144002) from peripheral blood samples of the participants in this study. 1×10 6 PBMCs were isolated from each milliliter of peripheral blood. The total RNA of isolated 3×10 6 PBMCs were isolated by TRIzol (ThermoFisher, 15596026).

Real-time PCR
After total RNA was isolated from PBMCs, it was reverse transcribed into cDNA. Real-time quantitative PCR was performed to quantify chemokine receptors, transcription factor and GAPDH levels by using SYBR Premix Ex Taq (TaKaRa, RR820A). Each reaction tube contained 20μL of reaction mixture, including 10μL of SYBR Premix Ex Taq, 1μL of each 10μM primer, 1μL of cDNA template and 7μL of ddH 2 O. The program was performed as follows: denaturation at 95°C for 10 min, followed by 40 cycles of 15 s of denaturation at 95°C and 60 s annealing/extension at 56°C. The comparative Ct method was used to calculate the relative abundance of different genes as compared with the expression of GAPDH. The primer sequences were listed in Table 2.
Enzyme linked immunosorbent assay (ELISA) The concentrations of chemokines, MCP-1, interferon-inducible protein-10 (IP-10), Interleukin 8 (IL-8) and interferon β (IFN-β) in the serum of COVID-19 patients and healthy controls were determined by ELISA kit according to the manufacturer's instruction (1117392, 1110802, 1117452, Dakewei. Inc, KE00187, Sanying. Inc.). The undiluted serum samples were added to the pre-coated ELISA plate, incubated for 2 h, and enzyme labeled antibody was then added. After washing the plate for 5 times, the substrate was added for color development, the absorbance was then determined using Epoch Microplate Spectrophotometer (Bio-Tek Instruments, Inc.) at 490 nm.

Statistical analysis
Analysis of Variance (ANOVA) tests were used to compare the plasma chemokine levels among the COVID-19 patients and healthy control groups. The Spearman rank correlation coe cient was used for linear correlation analysis between the expression level of plasma chemokine and IRF3. All data were analyzed using SPSS version 19.0 and GraphPad 5.0 software. P value less than 0.05 was considered statistically signi cant.

Results
MCP-1 level in mild COVID-19 patients is higher than that in healthy controls According to the basic information of severe COVID-19 and mild COVID-19 patients in Table 1, it was suggested that there were no remarkable differences between them from hospital time and some complications by statistically analysis. In order to further clarify the different role of chemokines in severe and mild COVID-19 patients, we detected three chemokine (including MCP-1, IP-10 and IL-8) in the serum among either severe, mild COVID-19 patients, or healthy controls. It was demonstrated that MCP-1 upregulation was observed in almost all COVID-19 patients no matter what is severe (296.7pg/mL±128pg/mL) or mild cases (215.9pg/mL±67.4pg/mL) than that of healthy control (36.2pg/mL±6.7pg/mL) (Fig. 1A) (P<0.01). IP-10 showed upregulation in severe COVID-19 patients (199.2pg/mL±82.6pg/mL) while no difference in mild patients (51.7pg/mL±39.4pg/mL) than that of healthy control (37.1pg/mL±3.1pg/mL) (Fig. 1B). Meanwhile, severe COVID-19 patients also showed the upregulation of IL-8 (51.3pg/mL±12.4pg/mL vs 27.2pg/mL±5.4pg/mL in healthy controls) (Fig. 1C). Recent reports indicated that MCP-1, IP-10, and IL-8 levels were higher in COVID-19 patients and even higher among those admitted to ICU 10 . We also found that the level of expression of the three chemokines was increased in severe COVID-19 patients. It has been suggested that chemokines play an important role in patients with severe COVID-19. The upregulation of MCP-1 in mild COVID-19 suggested that it may play the role in pathogenesis of mild COVID-19.

CCR2 show greater expression in PBMC from mild COVID-19 patients
Chemokines, when bind with corresponding receptors, play a chemotactic role in immune cells. In order to further clarify the role of chemokine in mild COVID-19 diseases, we evaluated the expression level of the receptors for MCP-1, IP-10, and IL-8, respectively. The expression level of the receptor of MCP-1, C-C motif receptor 2 (CCR2); the receptor of IP-10, chemokine (C-X-C motif) receptor 3 (CXCR3); and the receptor of IL-8, CXCR2 was assessed in the PBMCs from COVID-19 patients and healthy controls. We observed the upregulation of CCR2 (5.28±1.89) in mild COVID-19 patients than that in healthy controls (1.9±0.57) (P <0.01), while there was no difference between severe patients (1.83±0.43) and healthy controls ( Fig. 2A).

Higher expression of MCP-1 in mild COVID-19 patients is negative correlated with interferon regulator factors 3
Transcriptional activation of interferon regulator factors (IRFs) results in the launch of general antiviral programs. We then explored the expression level of IRF3, an important gene in the interferon signaling pathway, in mild COVID-19 patients. The expression of IRF3 was down-regulated (0.67±0.35 in mild COVID-19 patients vs 1.12±0.21 in healthy controls) (Fig. 3A) (P<0.01). Meanwhile, the downregulation of IFN-β was observed in mild COVID-19 patients (31.6±3.7 in mild COVID-19 patients vs 47.3±6.9 in healthy controls) (Fig. 3B) (P<0.01). To clarify the relationship between serum MCP-1 and expression level of IRF3, we performed Spearman rank correlation analysis using SPSS software, the results showed that IRF3 downregulation was signi cantly negative correlated with the level of MCP-1 (Fig. 3C) (P<0.01, r 2 =0.861). Our results suggest that MCP-1 may be an effective index for mild COVID-19 patients and higher expression of MCP-1 in mild COVID-19 patients may be correlated with the inhibition of IFN signaling.

Discussion
Previous studies have shown that elevated levels of pro-in ammatory cytokines, such as IFN-γ, TNF-α, IL-6 and IL-8, are associated with severe lung injury and adverse outcomes of SARS-CoV or MERS-CoV infection [18][19][20] . It has also demonstrated that severe COVID-19 patients have higher concentrations of chemokines in the serum than mild cases, suggesting that the magnitude of cytokine storm is associated with the disease severity 6,7 .
Most of COVID-19 patients in Shiyan city are mild cases 4 . In order to further evaluate the level of chemokines in mild COVID-19 patients, we detected the level of chemokines in the serum of mild COVID-19 patients admitted to Xiyuan Hospital and Renmin Hospital in Shiyan City. We selected monocyte chemokine, MCP-1, interferon induced protein 10, IP-10 and neutrophil chemokine, IL-8. MCP-1, this protein belongs to the C-C chemokine family and is a powerful monocyte chemotactic factor that is constitutively produced or induced by oxidative stress, cytokines, or growth factors. Monocytes and macrophages are the main source of MCP-1, which regulates the migration and in ltration of monocytes, memory T cells, and NK cells 21 . Huang et al. found that MCP-1 levels were higher in patients with COVID-19 and even higher among those admitted to ICU 10 24 . Therefore, monitoring the MCP-1 level early and intervening it may be a strategy to prevent the transformation from mild COVID-19 to severe COVID-19. IP-10 was initially identi ed as a chemokine whose secretion is induced by IFN-γ. IP-10 is secreted by neutrophils, endothelial cells, keratinocytes, broblasts, dendritic cells, astrocytes, and hepatocytes. Through its binding to chemokine receptor 3 (CXCR3), it regulates immune system responses by activating and recruiting leukocytes, including T cells, monocytes, and NK cells 25 . Therefore, IP-10 and CXCR3 play a key role in recruiting leukocytes to in amed tissues and in perpetuating in ammation, thereby making a major contribution to tissue damage 25 . Further comparison between ICU and non-ICU COVID-19 patients showed that plasma concentrations of IP10 were higher in ICU patients than non-ICU patients 10 , suggesting a relationship of IP10 with disease severity of COVID-19. Liu et al. associated elevated serum IP-10 levels with a higher viral load and greater lung damage in patients with SARS-CoV-2 26 . Recent reports suggested that the expression level of IL-8 was higher in patients with severe COVID-19 10 . Our results also proved that MCP-1, IP-10 and IL-8 were up-regulated in severe COVID-19 patients (Fig.1). It has been suggested that chemokines play an important role in patients with severe COVID-19. Meanwhile, MCP-1 has shown higher expression in patients with mild cases of COVID-19 (Fig.  1A). IP-10 and IL-8 showed upregulation in severe COVID-19 patients while no upregulation in mild patients (Fig. 1B, 1C). We further detected the receptors for MCP-1, IP-10, and IL-8 27 , respectively. We observed the upregulation of CCR2 in mild COVID-19 patients than that in healthy controls, while there was no difference between severe patients and healthy controls (Fig. 2). It was demonstrated that MCP-1 may participate in the pathogenesis of mild COVID-19 diseases.
Engagement of virus-speci c RNA structures culminates in oligomerization of these receptors and activation of downstream transcription factors, most notably IRFs and nuclear factor κB (NF-κB). We found that, in mild COVID-19 patients with higher level of MCP-1, the expression of IRF3, an important gene in the interferon signaling pathway, was down-regulated (Fig. 3A). There was no signi cant difference in the expression of NF-κB between mild COVID-19 patients and healthy controls (data not shown). IFN-β levels in serum of peripheral blood from mild COVID-19 patients were lower than that of healthy controls (Fig.3B). Meanwhile, IRF3 downregulation was signi cantly negative correlated with the level of MCP-1 (Fig. 3C). We also evaluated the expression level of IRF3 in the severe COVID-19 patients, but there was no relationship between MCP-1 and the expression of IRF3 (data not shown). So we regarded that the higher expression of MCP-1 in mild COVID-19 patients may be correlated with the inhibition of IFN signaling. Michael J, et al 28 demonstrated that IFN-β can induce MCP-1 transcription in bone marrow derived macrophages (BMDMs). Our results demonstrated a negative correlation of the level of MCP-1 in peripheral blood with the level of IRF3 expressed in PBMCs from mild COVID-19 patients in Shiyan. Thus we put forward a suggestion that IFN should be used to treat mild COVID-19 patients after they were detected as the upregulation of MCP-1 and downregulation of IRF3.
Other studies have already established that SARS-CoV-2 has greater sensitivity to type I IFN than SARS-CoV. Pre-treatment with IFN-α or IFN-β drastically reduced viral titers. These ndings also suggest that type I IFN may be effective as a prophylactic agent or an early treatment option for SARS-CoV-2. However, delayed IFN administration was of no bene t over a placebo 29 . Channappavanar et al. showed that delayed type I IFN expression can be detrimental in mice in the context of SARS-CoV-1 infection 30 . The timing of interferon exposition may be critical for controlling the virus and avoiding immunopathogenesis. Our results suggested that the serum IFN-β levels of mild COVID-19 patients were lower than that of healthy controls, which indirectly proved the effectiveness of early IFN treatment for COVID-19.

Conclusions
In summary, we found that higher expression of MCP-1 in mild COVID-19 patients may be correlated with the inhibition of IFN signaling. The nding adds our understanding to the immune-pathologic mechanisms of SARS-CoV-2 infection, and provides potential therapeutic targets and strategies.

Declarations
Ethics approval and consent to participate: The study protocol received approval from the Clinical Ethics Committee of Hubei University of Medicine (2020-TH-017). All individuals gave their informed consent to participate.
Consent for publication: Not applicable.
Availability of data and materials: All data generated or analyzed during this study are included in this published article.