Characterisation at the organ level of SARS-CoV-2-induced macrophage-dependent inflammation in the spleen
Lead Research Organisation:
University of Leicester
Department Name: Genetics
Abstract
This project aims to test if the spleen is a significant source of virus and inflammatory mediators during COVID-19. The induction of a cytokine storm is the cause of pathogenic inflammation both in SARS and COVID-19. Infection of splenic CD169+ macrophages by SARS-CoV-2 has been proposed to contribute to viral spread and excessive inflammation through pro-inflammatory cell death (Park Nat Rev Immunol 2020; Feng BioRxif 2020). Our interdisciplinary team has unique expertise with splenic CD169+ macrophages as key players during systemic infection (Ercoli NatMicrobiol 2018; Chung ALTEX 2019), and with use of a human spleen ex vivo perfusion model that has been authorised for work on COVID-19 (REC 18/EM/0057). We have now confirmed that a subpopulation of human splenic CD169+ tissue macrophages express both the SARS-CoV-2 receptor ACE2 and the spike-modifying protease TMPRSS2. We now propose to exploit our whole organ ex vivo human spleen perfusion model to characterise the steps in the early phases of systemic infection. This set up allows for detailed analysis of the infectious process over time. We will test the hypothesis that these CD169+ tissue macrophages serve as a hub for systemic spread of the virus. The main outputs will be the definition of key events during tissue macrophage infection by SARS-CoV-2 in the spleen. These outputs will provide important insights into the disease process and for optimising systemic host directed treatment strategies.
| Description | The main goal of the project was to set up an extracorporeal perfusion of human spleens to keep them functionally active for six hours in order to test if the SARS-CoV-2 virus would infect the immune cells; and to determine if there was a strong cytokine activation which is often fatal during COVID infection. We have successfully set-up and validated a human spleen perfusion model in a biosafety 3 lab utilising the Kidney Assist perfusion apparatus (XVIVO, The Netherlands). For the validation of the model, we utilised three human spleens sourced through our clinical trial at the Leicester University Hospitals NHS Trust (REC 18/EM/0057; ClinicalTrials gov NCT04620824). Monitoring of organs throughout the six-hour perfusion showed stable blood gas parameters and stable histology. Very unfortunately the situation in the hospitals worsened over the autumn and winter of 2021-22 due to the resurgence in COVID19 numbers with many elective surgeries delayed and then cancelled due to the scarcity of high dependency unit beds. This meant that we did not have enough suitable organs during the 18 months of the project to proceed to do infections with the SARS-CoV-2 virus. Still our ability to set-up the model and validate the quality of the organs has allowed us to set up new collaborations and has inspired colleagues to ask for ethical clearance to set-up the same model in other Universities in the UK, Italy, and China. During the project the Research Associate isolated 4 variants of SARS-CoV-2 (B.1.1.369, Alpha, Delta and Omicron) from diagnostic samples provided by our colleagues at the University Hospitals of Leicester. He has also further optimised plaque assay and focus forming assay methodologies and made the bulk stocks of virus required for the perfusion work. Furthermore, we have optimised SARS-CoV-2 infection of a human cell line Calu-3, to provide simple and plentiful hACE2/hTMPRSS2 positive cell controls for the microscopic imaging. From the three control spleens, we also harvested macrophages which allowed us to set up primary splenocyte macrophage cultures where we could document the expression of the SARS-CoV-2 receptor hACE2 and the protease hTMPRSS2 on a subset of primary splenic macrophages. The unfortunate fact that during the COVID pandemic the NHS saw a huge decrease in surgical treatment for cancer massively hampered our primary intent to develop the ex vivo human spleen perfusion model in the biosafety 3 lab and prevented us from monitoring events during early SARS-CoV-2 infection of spleen. However, we consider the opportunity to build novel collaborations and the fact that at least three different colleagues in the UK and overseas are setting up the same perfusion model is a great success and has contributed to the advancement of science. |
| Exploitation Route | The set-up and validation of the perfusion model is an important achievement with implications not only for COVID research but for infectious disease research in general. The three labs in the UK, Italy and China which have chosen to apply for, and in part already received, ethical clearance to set-up human organ perfusion models to study infection have applied with the intent to study other pathogens including the bacteria Staphylococcus aureus and Klebsiella pneumoniae and the malaria parasite. We view this as this as a great success and a contribution to the advancement of science. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Translational experimental models to study invasive infections in humans are scarce with 3D cell culture systems and organoid models receiving much attention currently. Still these models can not reproduce the complexity of the microanatomy and cell multiplicity of the natural organs. Having set up a organ infection model and presented this finding to colleagues had the very tangible impact that three international groups have started to set-up the same model to study infection. Colleagues in the UK, Italy and China have chosen to apply for ethical clearance to set-up human organ perfusion models to study infection. |
| First Year Of Impact | 2022 |
| Sector | Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | INF-ACT One Health Basic and Translational Research Actions addressing Unmet Needs on Emerging Infectious Diseases |
| Amount | € 114,500,000 (EUR) |
| Funding ID | PE0000007 |
| Organisation | Italian Ministry of Education, Universities and Research |
| Sector | Public |
| Country | Italy |
| Start | 11/2022 |
| End | 10/2025 |
| Title | Human organ ex vivo perfusion to study early events in infection |
| Description | Our pioneering work in porcine organs (Nat Microbiol 2018, Wanford Lancet Microbe 2021) paved the way to obtain ethical approval in the UK to use human spleens to study the early phase of infection (ClinicalTrials NCT04620824 and NCT05255042). Human spleens and liver segments are perfused for six hours with a synthetic oxygen carrier and infected with bacteria. fate of bacteria in the organ and perfusate are monitored by microscopy, and bacteriological methods. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2023 |
| Provided To Others? | Yes |
| Impact | Based on the initial data form our lab and follwoing disscussion and initiation of collaborations the model of ex vivo normothermic organ perfusion to study infection is being set-up by Jing-Ren Zhang at the Tsinghua University in Beijing China, by Matteo Ravaioli at the University of Bologna in Bologna Italy and by Wiebke Nahrendorf at the University of Edinburgh UK. I consider this a great impact. |
| Description | Ex-vivo human organ Perfusion to study the molecular and cellular parameters determining susceptibility to systemic Infection in Cirrhosis |
| Organisation | University of Bologna |
| Country | Italy |
| Sector | Academic/University |
| PI Contribution | Prof Ravaioli has applied for funding in Italy to develop the perfusion model set up at Leicester also in Bologna. Our group was added as coinvestigator on the project. |
| Collaborator Contribution | In the UK, we have two clinical trials, one on spleen perfusion and one on liver segment perfusion since the organs derive from surgery. In Italy the organs will derive from transplants and thus whole livers will be available to study the early events of infection. having whole organs will allow to extend perfusion form 6h to 24h, which is a very important melioration of the model. |
| Impact | Any output will be interdisciplinary as always microbiologists, immunologists and surgeons will eb involved in the work, but there are no published outputs so far. |
| Start Year | 2022 |
| Title | Tissue Models for Invasive Disease (TIMID) |
| Description | Our work on phase variation made us discover within-macrophage replication of bacteria and we have published this for mice and pigs. This led us to apply for a trial to explore fate of bacteria in the human spleen during the early stages of infection. This trial has been granted and work commenced in summer 2018. Study Title: Tissue Models for Invasive Disease (TIMID) Chief Investigator: Marco R Oggioni Site: University Hospitals of Leicester NHS Trust REC reference: 18/EM/0057 UoL Ref: UOL 0640 IRAS project ID: 219992 End date: 28/02/2021 |
| Type | Therapeutic Intervention - Vaccines |
| Current Stage Of Development | Initial development |
| Year Development Stage Completed | 2018 |
| Development Status | Under active development/distribution |
| Clinical Trial? | Yes |
| Impact | Work on the phase variation of S. pneumoniae during infection made us discover the intracellular phase of these bacteria. This led to the writing of the TIMID clinical trial. With this unique set up we plan to apply for new funding from the MRC in May 2019. |
| Title | Tissue Models for Liver Disease (TIMOLD); REC: 21/PR/0287 |
| Description | Based on our successful work of ex vivo perfusion of human spleens, we have applied and were granted by the HRA/REC authorisation for perfusion of human liver segments. This is the first model for ex vivo liver segment perfusion. As for our work with the human spleen, also here we will test the early events during infection. |
| Type | Management of Diseases and Conditions |
| Current Stage Of Development | Refinement. Non-clinical |
| Year Development Stage Completed | 2022 |
| Development Status | Under active development/distribution |
| Impact | Currently we are in the validation phase of the methodology. |