key: cord-0825733-zwftmuj3 authors: Zhou, Yonggang; Fu, Binqing; Zheng, Xiaohu; Wang, Dongsheng; Zhao, Changcheng; qi, Yingjie; Sun, Rui; Tian, Zhigang; Xu, Xiaoling; Wei, Haiming title: Pathogenic T cells and inflammatory monocytes incite inflammatory storm in severe COVID-19 patients date: 2020-03-13 journal: Natl Sci Rev DOI: 10.1093/nsr/nwaa041 sha: 0b703c00445f6c0bdd40d95f8e9dbd405a3a1e36 doc_id: 825733 cord_uid: zwftmuj3 nan Coronavirus, including SARS and MERS, has caused two large-scale pandemics in the last two decades 1, 2 . Although viral evasion of host immune responses and virus-induced cytopathic effects are believed to be critical in disease severity, studies from humans who died of SARS and animal models suggested that an excessive and aberrant host cytokine storm resulting in an exuberant immunopathology and lethal disease 9, 10, 11 . Inflammatory cytokine storm refers to the immune system gone awry and an excessive inflammatory response flaring out of control. Cytokine storms are associated with a wide variety of infectious and noninfectious diseases including graft-versus-host disease, autoimmune disease, severe virus infection, multiple organ dysfunction syndromes and chimeric antigen receptor (CAR)-T cell therapy 12, 13 . It has been reported that following SARS-CoV infection, dysregulated cytokine/chemokine responses and higher virus titers cause an inflammatory cytokine storm with lung immunopathological injury 12, 14 . Such Inflammation associated with the cytokine storm may begin at one local site but further spread throughout the body via the systemic circulation 12, 14 . Similarly, patients infected with SARS-CoV-2, that have been reported recently, have increased plasma concentrations of inflammation related cytokines, including interleukins (IL) 2, 7, and 10, granulocyte-colony stimulating factor (G-CSF), interferon--inducible protein 10 (IP10), monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1 alpha (MIP1A), and tumour necrosis factor  (TNF-), especially in moribund patients 15 . Importantly, COVID-19 patients have developed characteristic pulmonary ground glass changes on imaging and peripheral lymphocytes decreasing 14, 16, 17 . More importantly, a large number of inflammatory immune cell infiltrations were also found in a COVID-19 patient with pulmonary pathology 7, 8 . These phenomena suggest severe pulmonary inflammation and cytokine storm also exist in SARS-CoV-2 infection. At present, symptomatic treatments with organ support to moribund patients are the mainstays of clinical managements 17 . It is urgent to identify the immunopathology mechanism to delay the pulmonary immune injury. In patients infected with SARS-CoV, it has been reported that the severity of pulmonary immune injury correlated with extensive infiltration of neutrophils and macrophages in the lungs 18, 19 , accompanied with increased numbers of neutrophils and monocytes and lower CD8 + and CD4 + T cell counts in the peripheral blood samples 20, 21, 22 . To identify the immune characteristic of patients infected with SARS-CoV-2, peripheral blood samples from patients with severe pneumonia were collected for immune analysis. Consistent with previous clinical characteristics reports 23 2D , E). 41BB (CD137; TNFRS9) is an activation-induced co-stimulatory molecule, which is important to prime immune responses of cytotoxic CD8 + T cells 25 . In ICU patients infected with SARS-CoV-2, the expression of 41BB increased significantly compared to healthy controls ( Supplementary Fig. 2D, E) . It has been reported that co-expression of Tim-3 and PD-1 may represent a subset of T cells with more severe exhaustion in virus infections 26, 27 . It is worth noting that much higher percentage of co-expression Tim3 + PD-1 + T subset exist both in CD4 + and CD8 + T cells from COVID-19 patients ( Supplementary Fig. 2F -I), especially in ICU patients, suggesting the exhausted status in T cells in these patients infected To further identify the key pathogenic cytokines and the main source of these cytokines, interferon- (IFN-), TNF-, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-6 have been selected to be analyzed through intracellular cytokine staining, for these inflammatory mediators have been proven to be critical as the primary cause of inflammatory cytokine storm in patients infected with SARS-CoV or MERS-CoV 28, 29 . Without re-stimulation with PMA or incubation with monensin, high percentage of GM-CSF + and IL-6 + expressions could be found in CD4 + T cells from COVID-19 patients in both ICU and non-ICU patients compared to healthy controls (Fig. 1A, C) . ICU patients with more severe pneumonia showed correlated higher percentage of GM-CSF + and IL-6 + CD4 + T cells (Fig. 1A , C). Pathogenic Th1 cells with both IFN-γ and GM-CSF expression have been reported in central nervous system inflammation 30 . Importantly, aberrant pathogenic Th1 cells with co-expressing IFN- and GM-CSF exist only in ICU patients infected SARS-CoV-2, whereas little was found in non-ICU patients and healthy controls, indicating this pathogenic Th1 cells which have correlative evidence from patients with severe disease, play a critical role for hyper-inflammatory responses in SARS-CoV-2 pathogenesis (Fig. 1B, D) . Meanwhile, TNF- was not significantly up-regulated in CD4 + T cells from COVID-19 patients ( Supplementary Fig. 3A, B) . CD8 + T cells from ICU patients also showed higher expression of GM-CSF compared to those from non-ICU patients and healthy controls. IL-6 and TNF- were not found in CD8 + T cells ( Supplementary Fig. 3C, D) . Neither NK cells nor B cells were the secreting source of GM-CSF and IL-6 ( Supplementary Fig. 3E-H) . GM-CSF has been recently involved in the pathogenesis of inflammatory and autoimmune diseases, in a mechanism that controls diverse pathogenic capabilities of inflammatory myeloid cells. Among these myeloid cells, monocyte is the pathogenic GM-CSF responsive cells that require GM-CSF to initiate tissue damage in both mouse and human 31, 32 . To identify whether inflammatory monocyte exist in COVID-19 patients, phenotype and subpopulation of monocytes have been analysis. CD14 + CD16 + inflammatory monocyte subsets seldom exist in healthy controls. By contrast, significantly higher percentage of CD14 + CD16 + inflammatory monocyte exists in peripheral blood of COVID-19 patients. The percentage of CD14 + CD16 + monocyte was much higher in severe pulmonary syndrome patients from ICU ( Fig. 2A, C) . Moreover, these monocyte from COVID-19 patients also showed capability to secrete GM-CSF. Importantly, significantly higher expression of IL-6 secreted from these inflammatory monocyte especially in ICU patients, which let the inflammatory storm even worse (Fig. 2B, D) . Meanwhile, the number of GM-CSF + monocytes and IL-6 + monocytes increased rapidly (Fig. 2E) , suggesting the potential high risk of inflammatory cytokine storm caused by monocytes that may migrate to the lung and further develop into macrophage or monocyte derived dendritic cells. Thus, in COVID-19 patients, GM-CSF potentially links the severe pulmonary syndrome-initiating capacity of pathogenic Th1 cells (GM-CSF + IFN- + ) with the inflammatory signature of monocytes (CD14 + CD16 + with high expression of IL-6) and their progeny. These activated immune cells may enter the pulmonary circulation in large numbers and played an immune damaging role in severe pulmonary syndrome patients (Fig. 3 ). The study provides the detailed immunopathology report on SARS-CoV-2, suggesting excessive activated immune response caused by pathogenic GM-CSF + Th1 cells and inflammatory CD14 + CD16 + monocytes may connect pulmonary immunopathology leading to deleterious clinical manifestations and even acute mortality after SARS-CoV-2 infections. Consistent with the situation with SARS-CoV or MERS-CoV 14, 33 , it is remarkable that children always experience mild-moderate clinical illness, elderly individuals exhibit worse outcomes after infection with SARS-CoV-2, further indicating that mature excessive immune response towards these pathogenic human coronavirus infections play a key role in inducing severe pulmonary syndrome and even organ failure. Specific new drugs targeted SARS-CoV-2 may take a long time to evaluate and develop. At this critical moment, several marketed drugs to target inflammatory storm and reduce immunopathology could be considered 34 . Tocilizumab, that can specifically bind both membrane bound IL-6 receptor and soluble IL-6 receptor and inhibit signal transduction, is the first IL-6 blocking antibody approved for marketing and have proved its safety and effectiveness in therapy for rheumatoid arthritis 35 . In order to verify whether targeted IL-6 receptor and inflammatory signals, may potentially be the right way to save severe COVID-19 patients, we further launched the clinical trial using Tocilizumab to block the interleukin 6 receptor (ChiCTR2000029765). These severe patients recruited right now have inspiring clinical results including quickly decreased temperature and respiratory function improved. Many urgent questions remain to be answered. Evidence from alveolar washing fluid and organs autopsy from COVID-19 patients are further needed to verify whether and how these aberrant pathogenic immune cells play a fatal immune damage to cause organ functional disability and mortality. For details, see supplementary data. Supplementary data are available at NSR online. Representative density plots showing an analysis of GM-CSF and IL-6 expressions in gated CD45 + CD14 + monocyte cells isolated from peripheral blood in healthy controls, in ICU and non-ICU patients of COVID-19. (c) Statistics calculated by the percentage of CD14 + CD16 + subsets from monocytes. (d) Statistics calculated by the percentage of GM-CSF + or IL-6 + cells from CD14 + monocytes. (e) Statistics calculated by the cell number of GM-CSF + CD14 + or IL-6 + CD14 + monocytes. Data represent the mean ± SEM. One-way ANOVA. P<0.05 was considered statistically significant. Pathogenic CD4 + Th1 (GM-CSF + IFN- + ) cells were rapidly activated to produce GM-CSF and other inflammatory cytokines to form a cascade signature of inflammatory monocytes (CD14 + CD16 + with high expression of IL-6) and their progeny. These activated immune cells may enter the pulmonary circulation in large numbers and played an immune damaging role in severe pulmonary syndrome patients. The monoclonal antibodies that targets the GM-CSF or interleukin 6 receptor may potentially prevent or curb immunopathology caused by COVID-19. 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