key: cord-0870363-gts8jtae authors: Tan, Mingkai; Liu, Yanxia; Zhou, Ruiping; Deng, Xilong; Li, Fang; Liang, Kaiyan; Shi, Yaling title: Immunopathological characteristics of coronavirus disease 2019 cases in Guangzhou, China date: 2020-05-27 journal: Immunology DOI: 10.1111/imm.13223 sha: a619ef41b059a774e3420fdd20f0faa62cb62c48 doc_id: 870363 cord_uid: gts8jtae Coronavirus disease 2019 (COVID‐19) is a respiratory disorder caused by the highly contagious SARS‐CoV‐2. The immunopathological characteristics of COVID‐19 patients, either systemic or local, have not been thoroughly studied. In the present study, we analyzed both the changes in the number of various immune cell types as well as cytokines important for immune reactions and inflammation. Our data indicate that patients with severe COVID‐19 exhibited an overall decline of lymphocytes including CD4+ and CD8+ T cells, B cells, and NK cells. The number of immunosuppressive regulatory T cells was moderately increased in patients with mild COVID‐19. IL‐6, IL‐10, and C‐reactive protein were remarkably up‐regulated in patients with severe COVID‐19. In conclusion, our study shows that the comprehensive decrease of lymphocytes, the elevation of IL‐6, IL‐10, and C‐reactive protein are reliable indicators of severe COVID‐19. termed SARS-CoV-2 1 . This virus probably arose from an unidentified animal source and is subsequently transmitted from person to person 2 . Transmission of COVID-19 is through the air by coughing and sneezing, close personal contact, or touching a virus-contaminated object and then touching the mouth, nose, or possibly eyes. COVID-19's initial manifestations include cough, fever, and respiratory distress. The thorough clinical profile of COVID-19 has not been completely comprehended. Reported illnesses range from mild to severe or even death. The severity of COVID-19 varies among individual patients during the same outbreak and patient groups during different outbreaks in different regions. The immune response in COVID-19 patients, either systemic or local, has not been well studied even one month after the COVID-19 outbreak in China. Although the chest CT scan has suggested progressive pneumonia in COVID-19 patients, the inflammatory process and immune reaction have not been detailed. In one report, the elevation of the neutrophil number, serum IL-6, and Creactive protein (CRP) was found together with lymphopenia in COVID-19 patients 3 . Other clinical studies indicate that COVID-19 patients develop lymphopenia and high-levels of various cytokines such as G-CSF, IP-10, MCP-1, MIP-1A, and TNF-α 4, 5 . The surge of proinflammatory cytokines incurred the cytokine storm which might induce viral sepsis and tissue/organ damages to result in multiple organ failure. SARS-CoV-2-specific antibodies seem to be produced in COVID-19 patients, suggesting the mounting of humoral responses 3 . However, the comprehensive status of either innate immunity or adaptive immunity in COVID-19 patients remains largely unknown. In the current study, we analyzed multiple cytokines and immune cell populations in the blood of Chinese COVID-19 patients. Our study outlines the immunopathological profile in COVID-19 patients and may provide helpful information for developing future therapies against SARS-CoV-2 infection. This article is protected by copyright. All rights reserved The study was approved by the Ethics Committee of Guangzhou Eighth People's Hospital. Thirtyone individuals who were diagnosed to have mild/moderate COVID-19 symptoms (17 men, 14 women, average age=44.5), and 25 individuals who were diagnosed to show severe COVID-19 symptoms (18 males, 7 females, average age=66) in Guangzhou Eighth People's Hospital between January 2020 and February 2020 were enrolled in the study with informed consent. Diagnosis of COVID-19 infection, pneumonia, and clinical classification were based on the new coronavirus pneumonia diagnosis and treatment plan (trial version 6) issued by the National Health Committee of the People's Republic of China. The clinical classifications are on the basis of the following manifestations: (1) mild/moderate: fever, respiratory tract symptoms, and pneumonia on chest CT scan. Respiratory rate > 30 beats/min, or mean oxygen saturation < 93% (2) severe: one of the following: respiratory distress with respiratory rate > 30 beats/min, peripheral capillary oxygen saturation ≤ 93%, the ratio of the partial pressure of oxygen (PaO2) to the fraction of inspired oxygen (FiO2) < 300, respiratory failure requiring mechanical ventilation, Shock, ICU admission required for combined organ failure, pulmonary pathological progression. All patients were confirmed by the test on upper respiratory throat swab samples using the standard COVID-19 test kit. Individuals with fever and negative for the SARS-CoV-2 test were recruited as controls. Two to four milliliters of peripheral venous blood was withdrawn from each patient into K3 EDTA blood collection tubes. The complete blood cell count, lymphocyte count, and neutrophil count were determined on an XN-A1 automatic blood analyzer (Sysmex). The serum CRP levels were tested using a specific protein analyzer PA-990. The reagents, calibrators, and quality controls were from the same vendor. To measure lymphocytes and T cell subsets, 100 µl of whole blood was incubated in 900 µl of Tris-NH 4 Cl buffer (Thermo Fisher Scientific) at room temperature for 5 minutes to lyse erythrocytes. After two washes with phosphate-buffered saline (PBS), cells were incubated with BD Multitest 6-Color TBNK Reagent or BD Multitest CD3/CD8/CD45/CD4 (BD Biosciences) following the vendor's instructions. The BD Trucount™ Absolute Counting Tubes, which contain This article is protected by copyright. All rights reserved a known number of fluorescent beads, were used for quantifying leukocyte populations. After another two washes with PBS, cells were resuspended in 500 µl of PBS. To evaluate regulatory T cells, erythrocytes were lysed as described above. The blood samples were then incubated with FITC-conjugated CD4 antibody, Violet 450-conjugated CD127 antibody, PerCP-Cy5.5-conjugated CD45 antibody, and APC-conjugated CD25 antibody (5µg/ml each, all from BD Biosciences) for 15 minutes on ice. After another two washes with PBS, cells were resuspended in 500 µl of PBS. Cell samples were analyzed on a BD FACSCanto Plus flow cytometer. Among all collected events, single events were gated between FSC-A and FSC-H. Cell debris was excluded and intact cells were then gated from single events based on FSC-A and SSC. Each cell population was then detected based on the antibody staining. The data were assessed using the BD FACSDiva TM software. To analyze serum cytokines, the whole blood was centrifuged at 500×g for 5 minutes at 4 o C. The serum was carefully harvested and stored at -80 o C before analysis. Multiple serum cytokines were quantified using the Human Th1/Th2 Cytokine Kit II (Guangzhou Weimi Bio-Tech) following the manufacturer's manual. The principle of the cytokine tests was the same as the BD™ Cytometric Bead Array. The data were evaluated using the FCAP Array Software v3.0. The data were indicated as means ± standard deviation and measured by GraphPad Prism 6.0. The non-parametric Kruskal-Wallis test or One-way ANOVA with post-hoc Tukey HSD test was applied to compare the mean values or mean ranks among different groups, depending on the presence or absence of Gaussian distribution. Venous blood was collected from each patient at inpatient admission. These patients were suffering mild or severe COVID-19 symptoms and were not treated before blood collection. All cases were confirmed later by nucleic acid tests. To assess the general immune status of COVID-19 patients, we first quantified the absolute number of whole white blood cells (WBC) and immune cell populations in blood samples of patients at inpatient admission. As indicated in Figure 1A , the WBC number was significantly decreased in mild patients as compared with the control group. However, the WBC number was not remarkably changed in severe patients. The total lymphocyte number was decreased in severe patients but not in mild patients, suggesting the lymphopenia severe patients ( Figure 1B ). Neutrophils were increased in severe patients but not in mild patients ( Figure 1C ). The neutrophil-to-lymphocyte ratio (NLR), consistent with the change in lymphocytes, was moderately increased in mild patients and profoundly increased in severe patients, in comparison to the control group ( Figure 1D) . Notably, severe patients had a higher NLR than mild patients, suggesting that NLR was associated with the disease severity ( Figure 1D ). To analyze the adaptive immune cell populations, we conducted flow cytometry analysis on CD45+CD3+ total T cells, CD3+CD4+ T helper cells, and CD3+CD8+ cytotoxic T cells ( Figure 2A ). We found a similar decrease in CD45+ lymphocytes in COVID-19 patients ( Figure 2B ). Total T cell number was significantly decreased in COVID-19 patients, but no significant difference was observed between mild and severe patients ( Figure 2C ). Similarly, CD4+ T helper cells and CD8+ cytotoxic T cells were diminished in COVID-19 patients as compared with the control group, but no significant difference was found between mild and severe patients ( Figure 2D & 2E). The ratio between CD4+ T cells and CD8+ T cells was not changed ( Figure 2F ). revealed that only the frequency of CD4+ T cells was significantly reduced in severe patients ( Figure 2G to 2I) . Other immune cells including CD3-CD19+ B cells, CD3-CD16+CD56+ NK cells, and CD3+CD16+CD56+ NKT cells were also evaluated in blood samples ( Figure 3A ). We found that This article is protected by copyright. All rights reserved in comparison with the control group, there was a trend of decrease in B cell number in mild patients, and a notable reduction in B cells in severe patients ( Figure 3B) . A similar decrease was also seen in NK cells ( Figure 3C ). No remarkable change in the NKT number was observed ( Figure 3D ). Analysis of the frequencies of these cells in lymphocytes indicated that the frequency of B cells was significantly increased in severe patients ( Figure 3E ). But no profound change was found in the frequencies of NK cells and NKT cells ( Figure 3F & 3G) . Tregs are an important T cell subset for immune tolerance and anti-inflammatory reaction. In our study, we measured the number of CD3+CD4+CD25+CD127low T cells that are suggestive of the Treg-enriched population ( Figure 4A ). We found that the number of these cells were mildly increased in mild patients as compared with controls ( Figure 4B ). There was only a trend towards an increase in the number of this population in severe patients ( Figure 4B ). However, the frequency of these suggestive Tregs in lymphocytes was remarkably elevated in both mild and severe patients ( Figure 4C ). CD25 expression was enhanced in Tregs of severe patients, as demonstrated by their higher mean fluorescence ( Figure 4D ). Because CD25 is an IL-2 receptor subunit crucial for the survival, proliferation, and immunosuppressive activity of Tregs 6, 7 , the higher CD25 expression might indicate promoted Treg activity and function. However, Treg activity in severe patients should be investigated in the future. Figure 4B , IL-6 was significantly increased in severe patients but not in mild patients. Similarly, IL-10 was significantly increased in severe patients but not in mild patients ( Figure 4C ). CRP, a plasma protein synthesized by the liver as a sensitive marker of inflammation, was remarkably elevated in severe patients rather than mild patients ( Figure 4D ). However, we did not observe a significant correlation either between IL-6 and IL-10 or between IL-6 and CRP (Data not shown). This article is protected by copyright. All rights reserved The outbreak of COVID-19 poses an urgent demand for understanding the role of immunity in the progression of viral infection and subsequent pneumonia. As a new coronavirus, SARS-CoV-2 is highly contagious probably owing to the virus spread through asymptomatic-infected individuals 8 . Until recently, very few publications characterized the comprehensive changes in the innate and adaptive immunity in SARS-CoV-2 infected people, although some non-peer reviewed sources such as Medrxiv released some information. In the present clinical study, we analyzed almost all immune cell types in both mild and severe COVID-19 patients. We found lymphopenia consistent with former reports 4, 9, 10 . Neutrophils, however, were increased in severe patients. It is noteworthy that the lymphopenia seemed to arise from a comprehensive reduction of all lymphocyte populations, including CD4+ and CD8+ T cells, B cells, and NK cells. The decrease in blood lymphocytes might be due to the recruitment of reactive lymphocytes in the lungs. It will be helpful to investigate infiltrating cells in the lungs to test if the infiltrates are predominantly lymphocytes other than granulocytes. Another possibility is that lymphocytes are more sensitive than granulocytes to the disturbed homeostasis and then underwent significant cell death. Whatever the case, lymphopenia is an indicator of severe COVID-19. patients. This phenomenon might suggest the recruitment of reactive anti-viral CD8+ T cells into the infected tissues. According to reported T cell response in SARS-CoV infection, CD8+ T cell reaction was more frequent and robust than CD4+ T cell reaction 11 . It is, therefore, possible that this is also the case in COVID-19. Indeed, the latest case report shows the sign of excessive T cell In severe patients, the Treg number was comparable to that in control individuals. However, the This article is protected by copyright. All rights reserved Treg frequency in lymphocytes was remarkably increased in both mild and severe patients, Cytokines are produced by both innate immune cells and adaptive immune cells. IL-6 is a wellknown pro-inflammatory cytokine but it can also act as an anti-inflammatory mediator 13 . The cellular sources of IL-6 include myeloid cells, T cells, smooth muscle cells, endothelial cells, etc 14 . In COVID-19 patients, the dramatic increase in IL-6 likely originated from activated macrophages, consistent with another report 3 and similar to the IL-6 up-regulation in SARS patients 15 . IL-10 acts as an anti-inflammatory cytokine deriving from alternatively activated macrophages, Th2 cells, Tregs, etc 16 . The significant increase of IL-10 in severe patients might be negative feedback on the systemic and local inflammation. Surprisingly, IFN-γ remained at a low level in COVID-19 patients, because it is a crucial anti-viral cytokine produced by both CD4+ T cells, CD8+T cells, NK cells and macrophages 17, 18 , and it has been reported to participate in the cytokine storm in SARS patients 19 In conclusion, our study shows that the comprehensive decrease of lymphocytes and the elevation of IL-6, IL-10, and CRP are reliable indicators of severe COVID-19. Neutrophil-to-Lymphocyte ratio. *, p<0.05; **, p<0.01; ***, p<0.001. One-way ANOVA for comparing the mean difference. Immune responses in COVID-19 and potential vaccines: Lessons learned from SARS and MERS epidemic Covid-19: a puzzle with many missing pieces A pneumonia outbreak associated with a new coronavirus of probable bat origin Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China Pathological findings of COVID-19 associated with acute respiratory distress syndrome IL-2 induces in vivo suppression by CD4(+)CD25(+)Foxp3(+) regulatory T cells IL-2-and CD25-dependent immunoregulatory mechanisms in the homeostasis of T-cell subsets Transmission of 2019-nCoV Infection from an Asymptomatic Contact in Germany A new coronavirus associated with human respiratory disease in China Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China T cell responses to whole SARS coronavirus in humans Regulatory T cells in the treatment of disease The pro-and anti-inflammatory properties of the cytokine interleukin-6 Accepted Article This article is protected by copyright. All rights reserved 14 IL-6 in inflammation, immunity, and disease Up-regulation of IL-6 and TNF-alpha induced by SARS-coronavirus spike protein in murine macrophages via NF-kappaB pathway Regulation and functions of the IL-10 family of cytokines in inflammation and disease Direct Antiviral Mechanisms of Interferon-Gamma Sources of interferon-gamma (IFN-gamma) in early immune response to Listeria monocytogenes An interferon-gamma-related cytokine storm in SARS patients Osterhaus AD, Haagmans BL. Interferon-gamma and interleukin-4 downregulate expression of the SARS coronavirus receptor ACE2 in Vero E6 cells Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses Yaling Shi designed the study and wrote the paper. Mingkai Tan and Ruiping Zhou collected samples and performed most immune cell detection. Yanxia Liu and Xilong Deng conducted cytokine assays. Fang Li did the statistical analysis. Kaiyan Liang evaluated Tregs.The study is supported by the Guangzhou Science and Technology Program (Grant number: 202008010008).. The authors declare no conflict of interest. This article is protected by copyright. All rights reserved