key: cord-0300302-6jw7fdp2 authors: Albrich, Werner C.; Ghosh, Tarini Shankar; Ahearn-Ford, Sinead; Mikaeloff, Flora; Lunjani, Nonhlanhla; Forde, Brian; Suh, Noémie; Kleger, Gian-Reto; Pietsch, Urs; Frischknecht, Manuel; Garzoni, Christian; Forlenza, Rossella; Horgan, Mary; Sadlier, Corinna; Negro, Tommaso Rochat; Pugin, Jérôme; Wozniak, Hannah; Cerny, Andreas; Neogi, Ujjwal; O’Toole, Paul W.; O’Mahony, Liam title: Excessive inflammatory and metabolic responses to acute SARS-CoV-2 infection are associated with a distinct gut microbiota composition date: 2021-10-26 journal: bioRxiv DOI: 10.1101/2021.10.26.465865 sha: 6079e46674d0f1cf7c7f0e34f85ca0b1559aedaa doc_id: 300302 cord_uid: 6jw7fdp2 Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and associated clinical sequelae requires well-coordinated metabolic and immune responses that limit viral spread and promote recovery of damaged systems. In order to understand potential mechanisms and interactions that influence coronavirus disease 2019 (COVID-19) outcomes, we performed a multi-omics analysis on hospitalised COVID-19 patients and compared those with the most severe outcome (i.e. death) to those with severe non-fatal disease, or mild/moderate disease, that recovered. A distinct subset of 8 cytokines and 140 metabolites in sera identified those with a fatal outcome to infection. In addition, elevated levels of multiple pathobionts and lower levels of protective or anti-inflammatory microbes were observed in the faecal microbiome of those with the poorest clinical outcomes. Weighted gene correlation network analysis (WGCNA) identified modules that associated severity-associated cytokines with tryptophan metabolism, coagulation-linked fibrinopeptides, and bile acids with multiple pathobionts. In contrast, less severe clinical outcomes associated with clusters of anti-inflammatory microbes such as Bifidobacterium or Ruminococcus, short chain fatty acids (SCFAs) and IL-17A. Our study uncovered distinct mechanistic modules that link host and microbiome processes with fatal outcomes to SARS-CoV-2 infection. These features may be useful to identify at risk individuals, but also highlight a role for the microbiome in modifying hyperinflammatory responses to SARS-CoV-2 and other infectious agents. Given the substantial and significant differences in metabolite levels, we 159 examined in more detail the most significantly impacted pathways that associated 160 with COVID-19 severity (Fig. 3a) . Interestingly, levels of sulphonated bile acids were 161 particularly disrupted with disease severity. Host tryptophan metabolism was 162 associated with a heavy depletion of tryptophan, with enhanced generation of 163 kynurenate, kynurenine and quinolinate, at the expense of serotonin synthesis in 164 COVID-19 patients ( Fig. 3a and Supplementary Fig. 4a ). In contrast, microbial 165 tryptophan metabolites were present at lower levels in the serum of those with the 166 worst outcome ( Fig. 3a and Supplementary Fig. 4b ). Changes in circulating microbial 167 metabolites may be due in part to an impaired gut barrier (as indicated by increased 168 serum SCFA levels and lower citrulline levels, Supplementary Fig. 4c and 4d) , or 169 may reflect changes in the composition or metabolism of the gut microbiome. 170 Overall, metabolites associated with microbial metabolism (as described by Bar et 171 al 24 ) were significantly altered in those with severe disease and those with a fatal 172 outcome ( Supplementary Fig. 4e) . 173 Next, we performed a weighted co-expression network analysis restricted to reports and support the hypothesis that an overly aggressive immune response 280 contributes to immunopathology and severity 28, 29 . In addition to severity associated 281 factors, we have identified a subset of eight cytokines that are further dysregulated in 282 severe patients with a fatal outcome. Higher levels of IP-10 and IL-15 indicate 283 greater activation of a T helper 1 (Th1)-associated innate anti-viral response, while a 284 significant reduction in MDC levels may reflect the inhibitory effect of a Th1 285 environment on Th2 cytokines such as MDC. We were particularly interested in 286 TSLP as this cytokine is an epithelial cell-derived alarmin, which is released by 287 injured stromal cells to recruit and activate innate immune cells, and its blockade is 288 currently being investigated in asthma clinical studies 30,31,32 . In combination with the 289 chemokines MCP-1 and IL-8, and sICAM-1 (which modulates leukocyte adhesion 290 and migration across endothelial cells), elevated TSLP levels indicate a greater 291 amount of epithelial tissue damage and inflammatory cell recruitment to the 292 damaged sites in patients who do not recover from SARS-CoV-2 infection. As 1 4 SARS-CoV-2 is a lytic virus, it is possible that viral replication in epithelial cells may 294 directly drive TSLP levels in sera, although indirect effects on epithelial cells within 295 the respiratory tract or gut might also induce TSLP release. Importantly, TSLP levels 296 were previously shown to be elevated in patients with long COVID, suggesting that 297 long term impacts of SARS-CoV-2 on epithelial cells should be examined in more 298 detail, potentially guiding future therapeutic interventions 33 . 299 Significant metabolic reprogramming and compensatory responses are 300 evident in COVID-19 patients with severe disease and particularly in those with a 301 fatal outcome. Decreased serum levels of plasmalogens suggest a significant level 302 of systemic oxidative stress as these sacrificial phospholipids are preferentially 303 oxidised to protect more vulnerable membrane lipids such as polyunsaturated fatty 304 acids 34 . Altered tryptophan metabolism was particularly interesting to observe as the 305 profound shutdown in serotonin production coupled with accumulation of quinolinic 306 acid indicated a shift from production of neuroprotective compounds to production of 307 neurotoxic compounds, which might be clinically relevant 35 . An imbalance between 308 host and microbial tryptophan metabolism was also evident as serum kynurenine 309 levels increased, while products of bacterial tryptophan metabolism such as 310 indoleacetic acid were significantly decreased in those with severe and fatal 311 proinflammatory responses that cannot be contained by the regular feedback 344 mechanisms. While further studies will be required to determine causal interactions, 345 this study supports the hypothesis that successful responses to infectious agents 346 such as SARS-CoV-2 involve the gut microbiome mediated by effects on metabolism 347 and host inflammatory processes. 348 (≥18 years) patients who were admitted with Severe Acute Respiratory Syndrome 352 Coronavirus 2 (SARS-CoV-2) to four different hospitals in Switzerland and Ireland. From the Log2 transformed metabolomics data obtained from Metabolon, any 417 metabolite with no variance among samples was removed. Pairwise differential 418 abundance analysis was performed between conditions using R package LIMMA. 419 Benjamini-Hochberg correction (BH) was applied for each comparison. R packages 420 Boruta was applied for feature and tree number selection before random forest package was used to perform a hypergeometric test between list of significant 435 metabolites and reference. Importance plots, dot plots, bar plots, pca plots were 436 produced with R package ggplot2. Heatmaps were designed with the R package 437 ComplexeHeatmap. Networks were represented using Cytoscape 3.6.1 and 438 metabolites of interest highlighted. 439 For the microbiome analysis, the raw Illumina reads obtained for each sample 440 were quality-filtered using the trimmomatic program, using the default parameters 47 . 441 The quality filtered reads were then taxonomically classified using both DADA2 48 (for Clinical features of patients infected with 2019 novel coronavirus 680 in Wuhan Factors associated with COVID-19-related death using Autoantibodies against type I IFNs in patients with life-684 threatening COVID-19 Immunology of COVID-19: Mechanisms, clinical outcome, 686 diagnostics, and perspectives-A report of the European Academy of Allergy Immune response to SARS-CoV-2 and mechanisms of 689 immunopathological changes in COVID-19 Distribution of ACE2, CD147, CD26, and other SARS-CoV-691 2 associated molecules in tissues and immune cells in health and in asthma, COPD, 692 obesity, hypertension, and COVID-19 risk factors Large-Scale Multi-omic Analysis of COVID-19 Severity Plasma Markers of Disrupted Gut Permeability in Severe COVID-696 Imbalanced Host Response to SARS-CoV-2 Drives induced lung inflammation and injury in a murine model of lethal influenza infection Commensal microbiota 708 modulation of natural resistance to virus infection Gut microbiota composition reflects disease severity and 710 dysfunctional immune responses in patients with COVID-19 Distinct systemic and mucosal immune responses during acute SARS-CoV-2 infection Mechanisms 714 of microbe-immune system dialogue within the skin Obesity and disease severity magnify disturbed 717 microbiome-immune interactions in asthma patients Does the epithelial barrier hypothesis explain the increase in allergy, 719 autoimmunity and other chronic conditions? 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