Exploratory analysis of interleukin-38 in hospitalized COVID-19 patients.

INTRODUCTION: A major contributor to coronavirus disease 2019 (COVID-19) progression and severity is a dysregulated innate and adaptive immune response. Interleukin-38 (IL-38) is an IL-1 family member with broad anti-inflammatory properties, but thus far little is known about its role in viral infections. Recent studies have shown inconsistent results, as one study finding an increase in circulating IL-38 in COVID-19 patients in comparison to healthy controls, whereas two other studies report no differences in IL-38 concentrations. METHODS: Here, we present an exploratory, retrospective cohort study of circulating IL-38 concentrations in hospitalized COVID-19 patients admitted to two Dutch hospitals (discovery n = 148 and validation n = 184) and age- and sex-matched healthy subjects. Plasma IL-38 concentrations were measured by enzyme-linked immunosorbent assay, disease-related proteins by proximity extension assay, and clinical data were retrieved from hospital records. RESULTS: IL-38 concentrations were stable during hospitalization and similar to those of healthy control subjects. IL-38 was not associated with rates of intensive care unit admission or mortality. Only in men in the discovery cohort, IL-38 concentrations were positively correlated with hospitalization duration. A positive correlation between IL-38 and the inflammatory biomarker d-dimer was observed in men of the validation cohort. In women of the validation cohort, IL-38 concentrations correlated negatively with thrombocyte numbers. Furthermore, plasma IL-38 concentrations in the validation cohort correlated positively with TNF, TNFRSF9, IL-10Ra, neurotrophil 3, polymeric immunoglobulin receptor, CHL1, CD244, superoxide dismutase 2, and fatty acid binding protein 2, and negatively with SERPINA12 and cartilage oligomeric matrix protein. CONCLUSIONS: These data indicate that IL-38 is not associated with disease outcomes in hospitalized COVID-19 patients. However, moderate correlations between IL-38 concentrations and biomarkers of disease were identified in one of two cohorts. While we demonstrate that IL-38 concentrations are not indicative of COVID-19 severity, its anti-inflammatory effects may reduce COVID-19 severity and should be experimentally investigated.

Targeted proteomics identifies circulating biomarkers associated with active COVID-19 and post-COVID-19.

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic is caused by the highly infectious Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). There is an urgent need for biomarkers that will help in better stratification of patients and contribute to personalized treatments. We performed targeted proteomics using the Olink platform and systematically investigated protein concentrations in 350 hospitalized COVID-19 patients, 186 post-COVID-19 individuals, and 61 healthy individuals from 3 independent cohorts. Results revealed a signature of acute SARS-CoV-2 infection, which is represented by inflammatory biomarkers, chemokines and complement-related factors. Furthermore, the circulating proteome is still significantly affected in post-COVID-19 samples several weeks after infection. Post-COVID-19 individuals are characterized by upregulation of mediators of the tumor necrosis (TNF)-α signaling pathways and proteins related to transforming growth factor (TGF)-ß. In addition, the circulating proteome is able to differentiate between patients with different COVID-19 disease severities, and is associated with the time after infection. These results provide important insights into changes induced by SARS-CoV-2 infection at the proteomic level by integrating several cohorts to obtain a large disease spectrum, including variation in disease severity and time after infection. These findings could guide the development of host-directed therapy in COVID-19.

Plasma Oxylipins and Their Precursors Are Strongly Associated with COVID-19 Severity and with Immune Response Markers.

COVID-19 is characterised by a dysregulated immune response, that involves signalling lipids acting as mediators of the inflammatory process along the innate and adaptive phases. To promote understanding of the disease biochemistry and provide targets for intervention, we applied a range of LC-MS platforms to analyse over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). The second publication in a series reports the results of quantitative LC-MS/MS profiling of 63 small lipids including oxylipins, free fatty acids, and endocannabinoids. Compared to samples taken from ward patients, intensive care unit (ICU) patients had 2-4-fold lower levels of arachidonic acid (AA) and its cyclooxygenase-derived prostanoids, as well as lipoxygenase derivatives, exhibiting negative correlations with inflammation markers. The same derivatives showed 2-5-fold increases in recovering ward patients, in paired comparison to early hospitalisation. In contrast, ICU patients showed elevated levels of oxylipins derived from poly-unsaturated fatty acids (PUFA) by non-enzymatic peroxidation or activity of soluble epoxide hydrolase (sEH), and these oxylipins positively correlated with markers of macrophage activation. The deficiency in AA enzymatic products and the lack of elevated intermediates of pro-resolving mediating lipids may result from the preference of alternative metabolic conversions rather than diminished stores of PUFA precursors. Supporting this, ICU patients showed 2-to-11-fold higher levels of linoleic acid (LA) and the corresponding fatty acyl glycerols of AA and LA, all strongly correlated with multiple markers of excessive immune response. Our results suggest that the altered oxylipin metabolism disrupts the expected shift from innate immune response to resolution of inflammation.

Severe COVID-19 Is Characterised by Perturbations in Plasma Amines Correlated with Immune Response Markers, and Linked to Inflammation and Oxidative Stress.

The COVID-19 pandemic raised a need to characterise the biochemical response to SARS-CoV-2 infection and find biological markers to identify therapeutic targets. In support of these aims, we applied a range of LC-MS platforms to analyse over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). The first publication in a series reports the results of quantitative LC-MS/MS profiling of 56 amino acids and derivatives. A comparison between samples taken from ICU and ward patients revealed a notable increase in ten post-translationally modified amino acids that correlated with markers indicative of an excessive immune response: TNF-alpha, neutrophils, markers for macrophage, and leukocyte activation. Severe patients also had increased kynurenine, positively correlated with CRP and cytokines that induce its production. ICU and ward patients with high IL-6 showed decreased levels of 22 immune-supporting and anti-oxidative amino acids and derivatives (e.g., glutathione, GABA). These negatively correlated with CRP and IL-6 and positively correlated with markers indicative of adaptive immune activation. Including corresponding alterations in convalescing ward patients, the overall metabolic picture of severe COVID-19 reflected enhanced metabolic demands to maintain cell proliferation and redox balance, alongside increased inflammation and oxidative stress.

TRB sequences targeting ORF1a/b are associated with disease severity in hospitalized COVID-19 patients.

The potential protective or pathogenic role of the adaptive immune response to SARS-CoV-2 infection has been vigorously debated. While COVID-19 patients consistently generate a T lymphocyte response to SARS-CoV-2 antigens, evidence of significant immune dysregulation in these patients continues to accumulate. In this study, next generation sequencing of the T cell receptor beta chain (TRB) repertoire was conducted in hospitalized COVID-19 patients to determine if immunogenetic differences of the TRB repertoire contribute to disease course severity. Clustering of highly similar TRB CDR3 amino acid sequences across COVID-19 patients yielded 781 shared TRB sequences. The TRB sequences were then filtered for known associations with common diseases such as EBV and CMV. The remaining sequences were cross-referenced to a publicly accessible dataset that mapped COVID-19 specific TCRs to the SARS-CoV-2 genome. We identified 158 SARS-CoV-2 specific TRB sequences belonging to 134 clusters in our COVID-19 patients. Next, we investigated 113 SARS-CoV-2 specific clusters binding only one peptide target in relation to disease course. Distinct skewing of SARS-CoV-2 specific TRB sequences toward the nonstructural proteins (NSPs) encoded within ORF1a/b of the SARS-CoV-2 genome was observed in clusters associated with critical disease course when compared to COVID-19 clusters associated with a severe disease course. These data imply that T-lymphocyte reactivity towards peptides from NSPs of SARS-CoV-2 may not constitute an effective adaptive immune response and thus may negatively affect disease severity.

Extensive longitudinal immune profiling reveals sustained innate immune activation in COVID-19 patients with unfavorable outcome

COVID-19 differs substantially between individuals, ranging from mild to severe or even fatal. Heterogeneity in the immune response against SARS-COV-2 likely contributes to this. Therefore, we explored the temporal dynamics of key cellular and soluble mediators of innate and adaptive immune activation in relation to COVID-19 severity and progression. Forty-four patients with a PCR-proven diagnosis of COVID-19 were included. Extensive cellular (leukocytes and T-lymphocyte subsets) and serological immune profiling (cytokines, soluble cell surface molecules, and SARS-CoV-2 antibodies) was performed at hospital admission and every 3-4 days during hospitalization. Measurements and disease outcome were compared between patients with an unfavorable (IC admission and/or death) and favorable (all others) outcome. Patients with an unfavorable outcome had higher leukocyte numbers at baseline, mostly due to increased neutrophils, whereas lymphocyte and monocyte numbers were reduced. CRP, IL-6, CCL2, CXCL10, and GM-CSF levels were higher at baseline in the unfavorable group, whereas IL-7 levels were lower. SARS-CoV-2 antibodies were more frequently absent in the unfavorable group. Longitudinal analysis revealed delayed kinetics of activated CD4 and CD8 T-lymphocyte subsets in the unfavorable group. Furthermore, whereas CRP, IL-6, CXCL10, and GM-CSF declined in the favorable group, these cytokines declined with delayed kinetics, remained increased, or even increased further in the unfavorable group. Our data indicate a state of increased innate immune activation in COVID19-patients with an unfavorable outcome at hospital admission, which remained over time, as compared with patients with a favorable outcome.