key: cord-0714638-9qidtyux
authors: Li, Ang; Ling, Yun; Song, Zhigang; Cheng, Xiaobo; Ding, Longfei; Jiang, Rendi; Fu, Weihui; Liu, Yan; Hu, Huiliang; Yuan, Songhua; Chen, Jian; Zhu, Cuisong; Fan, Jun; Wang, Jing; Jin, Yanling; Zhang, Miaomiao; Zhu, Lingyan; Sun, Peng; Zhang, Linxia; Qin, Ran; Zhang, Wei; Qiu, Chenli; Shen, Yinzhong; Zhang, Lin; Shi, Zhengli; Zhao, Chen; Zhu, Tongyu; Lu, Hongzhou; Zhang, Xiaoyan; Xu, Jianqing
title: Early plasma IL-37 responses accompanied with low inflammatory cytokines correlate with benign clinical outcomes during SARS-CoV-2 infection
date: 2020-11-17
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa713
sha: 482f26a5fcb4eb3c426a8e8034ee27b1641e8338
doc_id: 714638
cord_uid: 9qidtyux

BACKGROUND: The immune protective mechanisms during SARS-CoV-2 infection remain to be deciphered for the development of an effective intervention approach. METHODS: We examined early responses of IL-37, a powerful anti-inflammatory cytokine, in 254 SARS-CoV-2-infected patients prior to any clinical intervention and determined its correlation with clinical prognosis. RESULTS: Our results demonstrated that SARS-CoV-2 infection causes elevation of plasma IL-37. Higher early IL-37 responses correlated with earlier viral RNA negative conversion, chest CT image improvement and cough relief, consequently resulted in earlier hospital discharge. Further assays showed that higher IL-37 was associated with lower IL-6 and IL-8 and higher IFN-α and facilitated biochemical homeostasis. Low IL-37 responses predicted severe clinical prognosis in combination with IL-8 and CRP. In addition, we observed that IL-37 administration was able to attenuate lung inflammation and alleviate respiratory tissue damage in human angiotensin-converting enzyme 2 (hACE2)-transgenic mice infected with SARS-CoV-2. CONCLUSIONS: Overall, we found that IL-37 plays a protective role by antagonizing inflammatory responses while retaining type I IFN, thereby maintaining the functionalities of vital organs. IL-37, IL-8 and CRP might be formulated as a precise prediction model for screening severe clinical cases and have good value in clinical practice.

M a n u s c r i p t 4

Coronavirus disease 2019 , caused by SARS-CoV-2 infection, poses an unprecedented challenge to global public health, and an effective treatment regimen remains elusive. Although the majority of COVID-19 patients have a benign clinical course, a fraction of them develop severe clinical complications that may even result in death [1] ; early alerting and identification of severe cases is critical and may allow for intensive medical care and prevent death [2, 3] . IL-37 is a recently identified member of IL-1 family. It was originally referred as IL-1F7 but renamed as IL-37 after identifying that it requires IL-18R receptor for its binding and biological activity [4] . As an anti-inflammatory cytokine, IL-37 could be produced by various cells including human peripheral blood mononuclear cells, macrophages, epithelial cells and activated B cells [5] ; Correspondingly, its receptor expresses on inflammatory cytokine secretory cells such as macrophages, mast cells and basophils [6] . There has been a growing body of evidence supporting that IL-37 plays an important role in a variety of inflammatory diseases [7] [8] [9] . In this study, we first identified a significant correlation between plasma IL-37 levels in COVID-19 patients and their clinical prognosis, with elevated plasma IL-37 likely suppressing inflammatory responses and thereby restraining the occurrence of cytokine storms. The absence of an IL-37 response may predict a severe clinical prognosis in SARS-CoV-2 infection.

All 254 patients were confirmed by SARS-CoV-2 viral RNA and admitted to the Shanghai Public Health Clinical Center (SPHCC), Shanghai, China, from February 6, 2020 to May 27, 2020 . Plasma samples from the 254 patients were collected upon their admission prior to treatment. According to the median level of IL-37, 127 COVID-19 patients with lower IL-37 levels (75 males and 52 females) and 127 patients (65 males and 62 females) with higher IL-A c c e p t e d M a n u s c r i p t 5 37 levels were enrolled. The distributions of their age, sex, other demographic information, biochemical and immunologic evaluations, and P values between the IL-37 low and IL-37 high groups are summarized in Table 1 . All patients were treated according to the Prevention and Control Plan, 4th edition (National Health Commission of the People's Republic of China, 2020).

Plasma IL-37 from the 254 COVID-19 patients was assayed by using a human IL-37/IL-1F7 ELISA kit (Beijing 4A Biotech Co., Ltd); measurement of 10 cytokines, including IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-22, and TNF-α was performed using the Simoa CorPlex human cytokine detection kit (Cat No: 85-0329, Quanterix, Billerica, MA, USA). All biosafety regulations were followed during the assays.

Clinical data for the 254 patients were collected from clinical test reports (blood routine, coagulation indexes, urine routine), biochemical reports (blood gas analysis, liver function, kidney function), immune reports (cytokine determination, absolute cell number determination, complement measurement) CT image reports, and vital sign monitoring (body temperature, respiration, blood oxygen saturation, blood pressure), among others ( Supplementary Figures 1 and 2 

A c c e p t e d M a n u s c r i p t 6 

The ACE2 transgenic mice used were 5-to 6-week-old ICR female mice, which were purchased from Hua-Fu-Kang, Inc. (Beijing, China), and split into two groups with 3 mice for each group. All mice were inoculated intranasally with 50 L (10 5 TCID 50 ) SARS-CoV-2 virus (provided by Dr. Zhengli Shi), experimental group were then administered intravenously with 12.5 g/kg recombinant human IL-37 [10] (purchased from Novoprotein Technology Co., LTD., Shanghai, China) at 12 and 48 hours post virus inoculation; Mice in control group were injected with PBS. The protocol was approved by Institutional Animal Care and Use Committee (IACUC) at Wuhan Institute of Virology, China Academy of Sciences, and implemented in Animal Bio-Safety Level 3 (ABSL-3) laboratory. All animal experiments were carried out in strict compliance with the "Guide for the Care and Use of Laboratory Animals" at Wuhan Institute of Virology, China Academy of Sciences to ensure the personnel safety and animal welfare.

Mice were sacrificed at 5 d.p.i and lung tissues were isolated and fixed with 4% formalin.

After sectioning by a microtome, slides were stained by hematoxylin and eosin (H&E) following standard protocol. The scanning procedures were conducted with TissueFAXS Confocal Plus 200 (TissueGnostics, Austrian) and the acquired images were analyzed by Strata Quest 6.0X software to calculate pathological score and inflammatory cell quantification.

Student's t test was used for parametric continuous data, and the unpaired two-sided Mann-Whitney U-test was utilized for nonparametric data. The log-rank (Mantel-Cox) test was used to evaluate significant differences between discharge rates, viral RNA negative conversion rates and cough relief rates of the two groups of patients. The significant P value cutoff was A c c e p t e d M a n u s c r i p t 7 set at 0.05. A predicted ROC curve of severely classified patients was examined, and the equation was calculated by SPSS 23 using binary logistic regression analysis. The python library "numpy-polyfit" was used to fit the smooth ROC curve. Quantitative indexes are described as mean values with the standard error of the mean (Mean±SEM), continuous features as medians (IQRs), and categorical features as numbers (%).

The cohort study was approved by the Ethics Committee of Shanghai Public Health Clinical Center (YJ-2020-S130-01), and all participants enrolled in this study signed informed consent forms. The animal protocol was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) at Wuhan Institute of Virology, China Academy of Sciences (WIVA05202009).

outcome.

An enzyme-linked immunosorbent assay was performed to quantify IL-37 levels in 254 COVID-19 patients at admission (prior to treatment intervention) and 36 healthy subjects (negative for SARS-CoV-2 RNA). We found that COVID-19 patients presented significantly higher plasma IL-37 levels (196.2±35.78, 95% CI, 125.7, 266.6) than healthy individuals Figure 1B) ; discharged rates within 9 days were 15.75% (20/127) and 1.57% (2/127), respectively ( Figure 1C ). All patients were discharged within 25 days for the IL-37 high group and 69 days for the low group (P<0.0001; log rank test) ( Figure 1D ).

We further examined the clinical prognoses of patients in the IL-37 high and low groups, including the timings and rates of SARS-CoV-2 viral RNA negative conversion, chest CT improvement, and cough relief. As expected, viral RNA negative conversion was significantly longer in the IL-37 low group (8.1±0.6 days, 95% CI, 6.88-9.28) than in the high group (4.5±0.36 days, 95% CI, 3.82-5.26) (P<0.0001), with 46% negative viral RNA in the IL-37 low group vs 74% in the high group within 6 days. Additionally, 31 days was required for all 122 patients in the IL-37 low group to become RNA negative, whereas only 20 days was needed for all 121 patients in the high group (P<0.0001; log rank test) ( Figure   2A ).

We next determined chest CT image improvement. Chest CT images of COVID-19 patients usually showed multiple small patch shadows and interstitial changes at the early stage and then gradually developed into multiple ground-glass opacities or infiltration shadows in both lungs [11, 12] . These lesions become absorbed and dissipate with the improvement of clinical symptoms and disease status [13] . Of 254 patients, 23 exhibited no manifestations on chest CT (10 in the IL-37 low group and 13 in the high group), 17 had only a single chest CT test report (10 in the IL-37 low group and 7 in the high group), and 18 showed long-term unchanged chest CT observations (9 in the IL-37 low group and 9 in the high group). After Overall, IL-37 responses may facilitate the retention of organ functions by antagonizing inflammation and thereby maintaining biochemical homeostasis.

We also assessed a panel of cytokines in the initial plasma upon patient admission, including IL-6, IL-8 and IFN-. Surprisingly, an imbalance of innate immune responses was noted with significantly higher IL-6 and IL-8 levels and lower IFN-levels in the IL-37 low group than in the high group (P <0.0001 for both IL-6 and IL-8, P =0.017 for IFN-). IL-6 and IL-8 are inflammatory cytokines and prone to causing immune pathogenesis, whereas IFN-exerts inhibitory activities against viral replication. Thus, the imbalanced innate responses in the IL-37 low group conferred a tendency of failure to restrain SARS-CoV-2 replication.

Accordingly, hypersensitivity CRP (HS-CRP) was also significantly higher in the low IL-37 group than in the high IL-37 group (P =0.049) (Figure 4A) .

A c c e p t e d M a n u s c r i p t 11 We next examined immune cell numbers in peripheral blood upon patient admission. Both myeloid and lymphoid-derived cells generally were in normal ranges, with abnormal levels in only a few patients, indicating that the majority of patients were at an early phase of the disease. Regardless, the IL-37 low group presented significantly lower CD8+ T cell and platelet counts than the high group (P=0.038 for CD8+ T cells and 0.043 for platelets, respectively), whereas no significant differences were observed for all other cells, including white blood cells (total cells of both myeloid and lymphoid-derived cells), monocytes, lymph cells, NK cells, CD3 + T cells and CD4 + T cells (Figure 4B and Figure 4C ). Other immunological indicators are shown in Supplementary Figure 2 .

Altogether, a low IL-37 response seems to attenuate host capacity to suppress inflammation and results in elevated inflammatory cytokine levels. Importantly, elevated IFN-was noted in the IL-37 high group, suggesting that IL-37 is unlikely to interfere with interferon responses.

Among the 254 cases, 20 were clinically sorted into the severe category. Interestingly, all 20 severe cases presented strongly low initial plasma IL-37 and elevated HS-CRP and IL-8 upon admission prior to any treatment ( Figure 5A) . Furthermore, significant reverse correlations were identified between IL-37 and HS-CRP (P =0.044) and between IL-37 and IL-8 (P =0.014) (Figure 5B) . The sensitivity of IL-37 alone to predict severe from moderate cases was 100%, with an AUC of 0.747 and specificity of 55.7%. To improve the prediction value, we added HS-CRP to IL-37 and observed that the AUC increased to 0.933, that the specificity reached 91.2%, but that the sensitivity decreased to 92.9% (Figure 5C Since IL-6 has been implicated to be involved in disease pathogenesis caused by SARS-CoV-2 infection, we examined its relationship with disease prognosis and with IL-37. As expected, plasma IL-6 in the severe group was significantly higher than that in the moderate group (Supplementary Figure 3A) , IL-6 levels were inversely correlated with serum IL-37 levels, high IL-37 producers were accompanied by low IL-6 levels (Supplementary Figure 3B) .

We also assessed the prediction value of IL-6, either alone or in combination with IL-37 or with both IL-37 and HS-CRP, for clinical outcomes of SARS-CoV-2 infection. As shown in Supplementary Figure 3C , the algorithm of triple combination of IL-6, IL-37, HS-CRP generated the best in disease predicting, followed by the IL-6 plus IL-37 combination and IL-6 alone. However, the predicting power of IL-6-IL-37-HS-CRP was still much less than that of IL-8-IL-37-HS-CRP ( Figure 5C ) Taken together, we identified a triple-marker of IL-8, IL-37, and HS-CRP to be remarkably predictive of clinical severity of SARS-CoV-2-causing diseases. Since all three evaluations can be assayed upon patient admission prior to treatment, this prediction model may possess great value in clinical practice for COVID-19 patients.

To further validate the anti-inflammatory ability of IL-37 during SARS-CoV-2 infection, we utilized a transgenic mice model that express human angiotensin-converting enzyme 2 (hACE2) and infected with SARS-CoV-2. After infection, the mice were split into two groups, one group received intravenous IL-37 (dissolved in phosphate buffer saline) administration at two time-points, 12h and 48h post infection whereas the other group was A c c e p t e d M a n u s c r i p t 13 administrated with phosphate buffer saline (PBS) as control. As expectedly, lung tissue histopathological examination revealed that more severe inflammatory cell infiltrations and tissue injuries were observed in control group, with more interstitial thickening and edema, blood capillary congestion and hemorrhage, exfoliation of pulmonary alveoli epithelium on day 5 after infection than the experimental group (Figure 6A and B) . To overall quantify the lung damage, we adopted pathological score and inflammatory cell quantification algorithm.

As shown, control group was scored significantly higher than IL-37 treatment group (P=0.006) (Figure 6C) , accordingly, more inflammatory cells per unit tissue area were found in control group (P=0.02) (Figure 6D ).

The COVID-19 pandemic due to SARS-CoV-2 infection has caused mass mortality worldwide and has become the most severe public health challenge; moreover, effective treatment for COVID-19 patients remains unavailable. Since hypercytokinemia and respiratory inflammation are known as critical pathogenesis mechanisms during respiratory viral infection [17] [18] [19] and IL-37 is a power suppressor against inflammation, we rationalized that IL-37 may play a protective role during SARS-CoV-2 infection and thereby should be explored for treatment. Here, we report that among 254 COVID-19 patients, early IL-37 responses prior to any treatment correlate with clinical outcomes. Elevated IL-37 responses probably favor balancing innate responses and thereby efficiently restrain viral replication to maintain the functionalities of host vital organs and thereby maintain internal homeostasis. In contrast, failure to mount IL-37 responses is likely to indicate a severe clinical prognosis.

Several studies have confirmed that the high inflammatory responses, presented as cytokine release syndrome (CRS) [20] , resulting from SARS-CoV-2 infection are among the preliminary pathogeneses for morbidity and mortality in COVID-19 patients [21] [22] [23] . In severe COVID-19 cases, dynamic IL-6 and IL-8 levels are associated with disease A c c e p t e d M a n u s c r i p t 14 progression [24] . Similarly, we identified elevation in IL-6 and IL-8 and their association with clinical pathogenesis. To antagonize inflammatory responses, corticosteroids are usually administered clinically; however, their efficacy in COVID-19-induced CRS is controversial, for it may increase viral replication and stimulate additional inflammatory responses. An ideal intervention is to retain intact innate immune responses but restrain inflammatory responses, and a novel treatment strategy with fine-tuning of the immune system is urgently needed [25] .

IL-37 is a newly discovered cytokine with strong anti-inflammatory activities. To date, researchers have focused more on its protective role and application in autoimmune diseases.

Studies showed that increased plasma IL-37 correlates significantly with ameliorated clinical manifestations in systemic lupus erythematosus [26] , in rheumatoid arthritis [27] and in inflammatory bowel disease [28] .

The unique anti-inflammatory mechanism may confer IL-37 with a wide range of antiinflammatory activities. Early researchers found that IL-37 can enter the nucleus and form a functional complex with Smad3 [9] , thereby reducing anti-inflammatory function by regulating gene transcription [29] . In recent years, a new anti-inflammatory mechanism involving binding to IL-18Ra was discovered. IL-37 is able to inhibit downstream proinflammatory signal kinases such as mTOR and MAPK. IL-37 also activates the antiinflammatory signaling molecules Mer and PTEN [7, 30, 31] .

The detailed mechanism of IL-37 against respiratory diseases has also been explored. Huang et al. [32] found that IL-37 may reduce airway inflammation by suppressing activation of NF-κB and STAT3 in asthma. Kim et al. [33] identified that IL-37 can inhibit TGF-b1-induced lung fibroblast proliferation, promoting the progression of idiopathic pulmonary fibrosis (IPF) disease. Qi et al. [34] observed that IL-37 attenuates secretion of inflammatory cytokines by downregulating the MAPK signal transduction pathway in an H1N1 infection A c c e p t e d M a n u s c r i p t 15 model, protecting mice from lung damage and improving survival. Zhou et al. [35] found that IL-37 induced by H3N2 infection is able to directly restrain the replication of influenza virus (IAV).

Here, we demonstrate that IL-37 is likely to play a protective role during SARS-CoV-2 infection. Early elevated plasma IL-37 prior to treatment correlates with low inflammatory cytokines, high IFN-a, a high number of immune cells and reduced hypoxemia. Furthermore, we demonstrated that IL-37 is able to effectively antagonize inflammatory responses in hACE2-transgenic mice after SARS-CoV-2 infection, the early administration of IL-37 resulted in reduction of the inflammatory cell infiltration, alleviation of lung tissue damage, thereby facilitated disease remission. Recently, Hadjadj et al. [36] found that exacerbated inflammatory responses and impaired type I IFN activity are hallmarks of severe illness, implicating the protective role of type I IFN. An important observation in our study is that the level of IFN-in patients with high plasma IL-37 levels was significantly higher than that in the IL-37 low group, suggesting that IL-37 is unlikely to suppress and even might enhance the production of IFN-α. These findings highlight the potential of IL-37 in the development of a novel therapeutic regimen, not only against coronaviruses such as SARS-CoV-2 and MERS but also against influenza and other respiratory viruses.

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SEM) (mm/h)

IQR) (mm/h)

ESR, erythrocyte sedimentation rate

We acknowledge all patients involved in the study. We acknowledge all the medical staff who provided clinical test data.A c c e p t e d M a n u s c r i p t

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t