key: cord-0959197-fa5tiaqo authors: Payne, Daniel J.; Dalal, Surita; Leach, Richard; Parker, Richard; Griffin, Stephen; McKimmie, Clive S.; Cook, Graham P.; Richards, Stephen J.; Hillmen, Peter; Munir, Talha; Arnold, Louise; Riley, Kathryn; McKinley, Claire; Place, Sandra; Baretto, Richard L.; Newton, Darren J. title: The CXCR6/CXCL16 axis links inflamm-aging to disease severity in COVID-19 patients date: 2021-01-25 journal: bioRxiv DOI: 10.1101/2021.01.25.428125 sha: 0a4bd7e5173cc0f6b30473beec39d17814af9de4 doc_id: 959197 cord_uid: fa5tiaqo Advancing age and chronic health conditions, significant risk factors for severe COVID-19, are associated with a pro-inflammatory state, termed inflamm-aging. CXCR6+ T cells are known to traffic to the lung and have been reported to increase with age. The ligand of CXCR6, CXCL16, is constitutively expressed in the lung and upregulated during inflammatory responses and the CXCR6/CXCL16 axis is associated with severe lung disease and pneumonia. Genome-wide association studies have also recently identified 3p21.31, encompassing the CXCR6 gene, as a susceptibility locus for severe COVID-19. We assessed numbers T cells expressing the chemokine receptor CXCR6 and plasma levels of CXCL16, in control and COVID-19 patients. Results demonstrated that circulating CD8+CXCR6+ T cells were significantly elevated with advancing age, yet virtually absent in patients with severe COVID-19. Peripheral levels of CXCL16 were significantly upregulated in severe COVID-19 patients compared to either mild COVID-19 patients or SARS-CoV-2 negative controls. This study supports a significant role of the CXCR6/CXCL16 axis in the immunopathogenesis of severe COVID-19. Coronavirus disease 2019 , caused by infection with severe acute 43 respiratory syndrome coronavirus-type 2 (SARS-CoV-2), encompasses clinical 44 phenotypes ranging from asymptomatic infection, through to severe disease and death. 45 The more severe end of this spectrum is often associated with respiratory pathology (1, 46 2). It is well established that the course of any infection is dependent on a number of 47 19 compared to controls (p<0.001 and p<0.0001 for absolute number and proportion 98 respectively). There was no significant difference in NK cells. 99 100 The receptor for SARS-CoV-2 is angiotensin converting enzyme 2 (ACE2) which is 101 highly expressed on alveolar epithelial type II cells of the lower respiratory tract (23). 102 Membrane bound CXCL16 is constitutively expressed on bronchial epithelial cells and 103 is released in metalloprotease-dependent manner in an inflammatory environment, 104 producing a soluble form which is chemotactic for CXCR6 + T-cells (24, 25) . The 105 CXCR6/CXCL16 axis mediates homing of T cells to the lungs in disease (26-28) and 106 hyper-expression is associated with localised cellular injury (29) (30) (31) with more severe lung pathology and poorer outcomes (36). We compared peripheral 117 blood T cell populations in severe and mild COVID-19 to control samples. This 118 revealed that absolute CD8 + CXCR6 + T cell populations were significantly reduced in 119 both severe and mild COVID-19 patients compared to controls (p<0.0001 and p<0.1 120 respectively; Figure 3e ), with significant reduction in absolute CD4 + CXCR6 + T cells 121 only between severe COVID-19 and controls (p<0.001; Figure 3g ). Strikingly, both 122 CD4 + and CD8 + CXCR6 + expressing T cells were present at extremely low proportions 123 in the blood of severe COVID-19 patients (n=12). inverse relationship between the concentration of blood CXCL16 and the proportion of 159 CD8 + and CD4 + CXCR6 + T cells in the blood in COVID-19 patients (Figures 4b and 160 4c). This suggests trafficking of CXCR6 + T cells to the lung drives a pro-inflammatory 161 immunopathology in severe COVID-19, with these cells infiltrating into the tissue, 162 which is supported by lower numbers of CD8 + T cells reported in broncho-alveolar 163 lavage in mild compared to severe COVID-19 patients (41). Furthermore, the 164 CXCR6/CXCL16 axis has been implicated in both infective (influenza) and non-165 infective (sarcoidosis) inflammatory lung diseases (25, 26). However, this inverse 166 relationship between CXCL16 levels and CXCR6 + T cells and may also be explained by 167 either CXCL16 binding to CXCR6 causing receptor internalisation, epitope masking or 168 CXCL16-mediated T cell apoptosis. 169 Following infection with SARS-CoV-2, there is potential for pre-existing inflammatory 170 CXCR6 + populations, associated with either co-morbidity and/or inflamm-aging, to be 171 recruited from the blood to the lungs mediated by CXCL16, resulting in more severe disease (42). Similarly, in other diseases characterised by a T cell infiltrate, such as type 173 1 diabetes, high expression of this chemokine receptor and ligand have been reported in 174 pancreatic tissue where they play a role in inflammation (43). 175 176 CD8 + CD161 ++ CXCR6 + T cells, have the capacity to be cytotoxic and express the 177 transcription factor RORĪ³t, which is associated with a Th17-like phenotype and a pro- Samples were analysed using 2 phenotyping panels (Supplementary Table 1 Plasma CXCL16 levels were measured in duplicate using a commercial ELISA 243 (ThermoFisher Scientific), with a coefficient of variation of less than 10%, according to 244 the manufacturer's specifications. 245 Data was analysed with GraphPad Prism version 9.0.0 (GraphPad software). 247 Categorical data was compared with Mann-Whitney statistical analyses. 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