Cell specific pro- and antiinflammatory lipidmediator shift in critically ill Covid19 patients

Introduction The COVID-19 pandemic had a significant impact on morbidity and mortality in Germany challenging intensive care unit (ICU) capacities across the country. To delineate the high variability in disease severity, clinical presentation and outcome, we focused on cellular regulators of inflammation and resolution on a single cell level to gain a deeper understanding of the patient's individual inflammatory response and their impact on survival. Methods Written informed consent was obtained from all patients and healthy controls. The study was approved by the local ethical review board (Az249/20 S-EB). To characterize the peripheral immune landscape, we performed a 14 parameter flow cytometric analysis of PBMCs of 32 critically ill CoV2 patients and a targeted HPLC-MS/MS of previously sorted PBMCs. All data was analyzed and correlated to clinical parameters and patients' outcomes (Fig. 4). Results As known [1], computational analysis of flow cytometry revealed a strong decrease of B Cell and CD8+ T Cell ratios and an increase of monocytes in critically ill CoV19 patients compared to control (Fig. 1A). Interestingly, non-survivors displayed an increased ratio of CD16+ monocytes and proinflammatory IL- 1beta in monocytes, B and T cells, while HLADR receptors were downregulated correlating with clinical outcome (Fig. 1B). Not unexpectedly, we saw a major increase in proinflammatory lipidmediators, such as PGJ2, PGF2, TxB2 (Fig. 1C). Additionally, our analysis revealed that not only the amount, but also the source of those mediators was shifted from CD16 to classical CD14 monocytes, even more pronounced in non-survivors. CD16 monocytes of CoV2 patients, however, lost the ability to generate proresolving lipidmediators depending on cytochrome p450 (Cyp450) or soluble epoxide hydrolase (sEH) TxB2 (Fig. 1D). Conclusions Even though a lot of insight into CoV2 has been gained over the last 2 years, relatively little is known about the impact of immune changes in critically ill patients. With this study, we are the first to attribute lipid mediators to specific cell types. Our findings show that TxB2 in critically ill CoV2 patients, which correlates with mortality in CoV2 [2], is produced mainly in CD14 monocytes. We further report that specifically non-survivors display increased ratios of non-classical CD16 monocytes, which are impaired to generate a major class of lipidmediators depending on Cyp450. In conclusion, these data provide evidence that not only the absolute amount of pro- and anti-inflammatory mediators, but also the cellular source of these mediators remains key to fully understand their role in critically ill CoV2 patients. (Figure Presented).

Immunological characterization of COVID-19 endotypes of critically ill patients

Introduction Over the last 20 months Sars-CoV-2 research revealed tremendous insights into the pathophysiology resulting in vaccines and first immunomodulatory therapies in an unprecedented short time [1]. However, the patient's individual clinical course is remarkably heterogenous and the effectivity of immunomo-dulatory therapies especially in critically ill patients is still very variable [2]. Therefore,a better understanding of the patients' individual immune response is needed [3]. Methods Unbiased machine learning grouped 323 Covid-19 patients treated at the Klinikum Rechts der Isar ICU into 3 different clusters. For this we queried the ICU electronic files and analyzed relevant clinical features (Fig. 1+2). To delineate biological differences within these clusters, we applied a 14 parameter flow cytometric panel to PBMCs from 27 of these CoV2 ICU patients. Flow data was analyzed using FlowJo and the FlowSOM package for unsupervised clustering (Fig. 3+4). Written informed consent was obtained from all patients and healthy controls. The study was approved by the local ethical review board (Az249/20 S-EB). Results Patients from cluster 1 had above ICU average respiratory function (Fig. 2), reduced liver function and received lower dose catecholamines. Immunologically these patients had significantly higher amount of CD3+CD4+ T helper cells (Fig. 5). Whilst B cell numbers were reduced, they were highly activated (HLA-DR-ordf). Activated monocytes produced high amounts of TNFa. Interestingly, proinflammatory CD14+ HLA-DRlow monocytes were not increased. Cluster 2 contains patients with renal impairment, an increased tendency for bacterial infection and elevated blood lactate levels. Cluster 3 is made up of long-term ICU patients with severely reduced respiratory function and high ECMO-dependency (Fig. 1). These patients had significantly increased ratios of activated innate immune cells. We have detected elevated levels of an interesting population of CD14+ HLA-DRlow monocytes, a well-established player of immune suppression [4], while cytotoxic T cells and B cells were found to be significantly reduced. Conclusion These data provided evidence that clinically defined endotypes of critically ill Covid-19 patients exhibit a distinct immune profile. The immunological differences support our theory that these endotypes might require personalized immunomodulatory therapies to restore the pro-regenerative cell function in ICU Covid-19 populations and improve patient outcome in the future.

Immune phenotypes correlate with the outcome of severe Covid-19 infection

Despite the tremendous impact of the Sars-CoV-2 global pandemic and becoming focus of scientific research[1], many aspects of the disease and its pathophysiology, especially concerning prognostic parameters and treatments remain uncertain. The aim of our study is to assess and link immune profiles of the dysregulated cellular immune response in patients hospitalized with severe Covid-19 to their outcomes. Therefore, we immune phenotyped severly ill Sars-CoV-2 patients on our ICU and to surviving to non-surviving patients and healthy controls. Methods Using flow cytometry (BD-Fortessa), we created a 14-parameter immunoprofile of 25 Covid-19 patients from our ICU and 11 of healthy control individuals. The analysis was based on live/dead control, CD3, CD4, CD8, CD19, CD66b, CD14, CD16, IL-10, TNF-α, IL-1β, HLA-DR and IL-6 antibodies. Both clusters (survivors, n=16;non-survivors, n=9) and healthy controls (n=11) were compared with each other by Kruskal-Wallis test with Dunns-Ls post-test correction for multiple testing (Prism V.9.0). The patients in both groups had a similar age and at the time of analysis (Fig. 1A), the treatment was insignificantly more invasive in non-survivors than in survivors (7-point WHO ordinal scale means 5.8 vs. 5.3, p = 0.43) (Fig.1 D). Similarly, the blood tests and the viral loads were comparable in both groups. Study has permission from the ethic commission (AZ-249/20 S-EB). Results The study showed that cell specific cytokine expressions are distinct in survivors compared to non-survivors even at an early stage of the critical disease. Surviving Covid patients showed increased TFNá levels throughout all cell populations, which met significance in CD4+ T (Fig.2 A) Cells and CD135+ DC. (Fig. 4 C). IL-6 levels, however, were significantly lower in CD4+ T cells of survivors (Fig. 2 B). Similarly, proinflammatory, classical CD16+ monocytes of non-survivors exhibited an increase in IL-6 and IL-1â. Moreover, dendritic cells of non-survivors seemed to be exhausted revealing less TNFá and IL-6 and IL-1â (Fig 4) Conclusion: Taken together, a disability of monocyte activation and exhaustion of dendritic cell reaction was associated with a worsened outcome of severely ill Covid-19 patients [2,3]. On the contrary a sufficient TNFá response, especially of CD4 and dendritic cells might be required to overcome the infection. Therefore, our findings suggest that measuring cell specific levels of cytokines and cell population shifts might be of high clinical relevance to predict the outcome of the disease and offer new therapeutical options for these patients.