The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in millions of deaths worldwide. While the virus primarily affects the respiratory system, the clinical course is highly variable and often unpredictable. A dysregulated immune response, characterized by high levels of pro-inflammatory cytokines, is considered a major driver of COVID-19 severity. However, factors contributing to this immune dysregulation remain incompletely understood. The human microbiota, particularly the gut and oropharyngeal microbiomes, plays a crucial role in immune system modulation through metabolite production. Previous studies have indicated altered microbiome composition in COVID-19 patients, and these changes might influence the immune response. Tryptophan, an essential amino acid, is metabolized by both the host and the microbiota into various metabolites with immunomodulatory functions. Alterations in tryptophan metabolism have been implicated in inflammatory diseases. This study hypothesizes that alterations in the microbiome and metabolome, particularly tryptophan metabolism, contribute to the variable clinical course and immune dysregulation observed in COVID-19.

Several studies have shown an association between COVID-19 severity and alterations in the gut and oropharyngeal microbiota. Reduced bacterial diversity and enrichment of opportunistic pathogens have been reported in severe cases. Elevated levels of inflammatory cytokines, such as IL-1β, IL-6, and CXCL8, have also been observed. Furthermore, the role of tryptophan metabolism in immune regulation is well-established. Tryptophan is metabolized through the kynurenine pathway and the serotonin pathway, producing metabolites with diverse effects on immune cells. Imbalances in these pathways have been linked to various inflammatory conditions. This study builds upon existing literature by integrating microbiome, metabolome, and immune response data to understand their interplay in COVID-19 pathogenesis.

This prospective cohort study included 30 hospitalized COVID-19 patients with varying disease severity and 15 uninfected controls. Repeated samples (plasma, stool, urine, oropharyngeal swabs, and in some cases tracheobronchial secretions and PBMCs) were collected. Shotgun metagenomic sequencing was performed on stool and oropharyngeal swabs to characterize the microbiota. Metabolomic analysis using LC-MS and GC-MS was conducted on plasma and urine samples to identify differences in metabolite profiles. Multiplex cytokine ELISA was used to measure plasma cytokine levels. Single-cell RNA sequencing (scRNA-seq) of PBMCs was performed to analyze immune cell populations and gene expression. An integrated statistical approach was used to analyze the omics data and clinical information, accounting for potential confounders such as antibiotic use and comorbidities. Specific methods included DADA2 pipeline for 16S rRNA gene analysis, Biocrates MxP Quant 500 assay and GC-MS analysis for metabolomics, and Seurat package for scRNA-seq data analysis. Statistical testing incorporated linear mixed-effect models and likelihood ratio tests to account for repeated sampling and potential confounders.

Severe COVID-19 was associated with decreased alpha diversity of the gut microbiota compared to controls and mild cases. Several potentially beneficial commensals, including *Faecalibacterium*, were depleted in severe COVID-19. Oropharyngeal microbiota disturbances were mainly linked to antibiotic use. Plasma levels of kynurenine were significantly higher in COVID-19 patients, particularly in severe cases, while levels of several other tryptophan metabolites, including tryptophan itself, were reduced. Reduced levels of various tryptophan metabolites, including 5-hydroxytryptophan and indole-3-propionic acid, correlated with *Faecalibacterium* depletion. Decreased tryptophan and increased kynurenine levels were associated with enhanced production of pro-inflammatory cytokines, such as IFNγ. Severe COVID-19 was also associated with reduced levels of lysophosphatidylcholines and secondary bile acids. Single-cell RNA sequencing revealed alterations in immune cell populations, with an increase in classical monocytes and a decrease in non-classical monocytes in severe COVID-19. Integrated analysis revealed correlated alterations across microbiome, metabolome, and immune response, suggesting a complex interplay among these factors in severe COVID-19 pathogenesis.

The findings of this study support the hypothesis that gut microbiota dysbiosis and altered tryptophan metabolism contribute to the dysregulated inflammatory response observed in severe COVID-19. The depletion of beneficial commensals, particularly *Faecalibacterium*, and the shift in tryptophan metabolism towards kynurenine production, may create a pro-inflammatory environment. The observed correlations between decreased tryptophan metabolites, increased kynurenine, and elevated pro-inflammatory cytokines suggest a potential mechanistic link. Furthermore, the alterations in lysophosphatidylcholines and secondary bile acids further support the disruption of metabolic homeostasis. These results highlight the potential for targeting the gut microbiome and tryptophan metabolism as therapeutic strategies for COVID-19. The interplay between microbiome, metabolome, and immune system needs further investigation to understand the exact mechanisms involved and develop targeted therapies.

This multi-omics study demonstrates a significant association between gut microbiota dysbiosis, altered tryptophan metabolism, and dysregulated immune responses in severe COVID-19. The findings highlight the interconnectedness of these factors and suggest potential therapeutic targets. Future research should focus on mechanistic studies to elucidate the causal relationships between these factors and explore the therapeutic potential of modulating the gut microbiome and tryptophan metabolism in COVID-19.

This study has several limitations. The sample size is relatively small and limited to a single center, limiting the generalizability of the findings. The inability to collect samples during the earliest phases of infection prevents a comprehensive understanding of early-stage mechanisms. Lack of dietary information might influence the interpretation of microbiome changes. The absence of an intensive care control group makes it challenging to fully disentangle critical disease and hospitalization effects from COVID-19-specific effects. Finally, limited respiratory microbiome data reduced the depth of analysis in this area.