4. Discussion
Our study emphasizes the impact of SARS-CoV-2 infection on the GI which might be associated with the onset of PCS Fatigue. We propose that the viral infection dysregulates immunological pathways activating the innate immune response and the GI barrier function as indicated by the detected intestinal low-grade inflammation and a GI barrier leakage. Further, we identified pre-existing GI complaints as a risk factor for developing PCS Fatigue.
4.1 Dysregulated innate immunity may predispose for GI barrier leakage in PCS Fatigue
Elucidating mechanisms of PCS involves identifying predisposing clinical phenotypes. Recent research revealed diverse auto-antibody specificities in adaptive immunity without a clear symptom correlation [7, 8]. In our study, SARS-CoV-2 convalescent individuals have significantly higher titers of IgA (Supplementary Figure 1 ). The evaluation provides limited evidence as additional factors, such as virus variant, type of vaccination and the order of experienced immunological events, were not considered in the analyses of the SARS-CoV-2-related antibody responses and cannot be accurately assessed in our small cohort sample sizes. Early during the pandemic, low IgM or IgG3 antibodies were described to be associated with a higher risk of PCS [18]. In terms of innate immunity, the immune response, particularly changes in monocytes, dendritic cells, and mannose-binding lectin (MBL), is less understood [19, 20]. It was shown that 10 months after COVID-19, convalescent patients had lower absolute counts of granulocytes, monocytes, and lymphocytes compared to controls [10]. Low MBL levels were linked to higher cytokine levels and PCS symptoms like severe Fatigue [21]. MBL deficiency may lead to excessive IL-6 production, contributing to long-term inflammation [22]. PCS patients in our study showed significantly higher IL-6 levels compared to healthy controls (Figure 2 ). However, these results should be interpreted carefully as the assay signals in the range of the LOD. More sensitive methods are recommended for future studies. PCS patients were already susceptible to infections before SARS-CoV-2 infection (Table 2 ), indicating a possible underlying dysregulation of the innate immune system. We also observed frequent pre-existing signs of hypermobility in PCS and ME/CFS patients. Conditions like Ehlers-Danlos Syndrome and Joint Hypermobility Syndrome, often associate with systemic issues, including respiratory problems and GI complaints [23, 24]. It remains unclear which immune phenotypes may predispose for developing PCS. Genetic and epigenetic studies have previously attempted to address this question [25-27].
4.2 Multi-organ symptoms during acute COVID-19 are associated with the onset of PCS Fatigue
We were not able to observe viral shedding indicated by SARS-CoV-2 RNA in saliva, blood or stool samples in our study (data not shown). SARS-CoV-2 fecal shedding typically lasts 17.2 days on average but can continue for several months [28]. During this time, SARS-CoV-2 is not detectable over a longer period, but PCS symptoms may persist [29]. Biopsy studies in patients with GI conditions, such as IBS, IBD, or gastroesophageal reflux disease, found SARS-CoV-2 RNA or antigen in GI mucosal tissues in 50 to 70% of patients, suggesting a viral reservoir [30, 31]. However, these results came from patients with pre-existing GI diseases, leaving open whether GI damage enabled viral persistence. Pre-existing barrier or immune impairments might hinder the elimination of residual virus reservoirs or infected cells. Viral persistence, whether from SARS-CoV-2 or reactivated latent viruses like EBV, may contribute to continuous inflammation. The multi-organ spread of SARS-CoV-2 during acute infection likely contributes significantly to PCS development by disrupting immune functions, leading to prolonged immune activation or dysregulation. Our data indicate that patients with multisystemic symptoms, including GI, respiratory, and neurocognitive complaints during acute COVID-19, are more likely to develop PCS (Supplementary Table 1 ). PCS patients more often had multi-organ symptoms during acute infection compared to those who recovered. These observations align with recent literature discussed in more detail elsewhere [32, 33].
4.3 GI barrier leakage predisposes for PCS Fatigue
We showed that PCS patients had significantly more often pre-existing GI complaints before acute infection (Table 2 ). Pre-existing food intolerances, but also with a lower frequency thyroid disease, were more common in PCS and ME/CFS patients. These conditions affect the bidirectional communication between the microbiome, its metabolites, and host epithelial tissue dysregulating the gut-brain axis. In line, patients suffering from post-infectious Fatigue syndromes exhibit a dysfunctional intestinal barrier, associated with distinct fecal microbial metagenomic profiles [34]. We showed that PCS patients suffered from a significantly higher LBP/sCD14 ratio when compared to all other groups (Figure 1 ). LBP is linked to bacterial wall components that breach the intestinal barrier and enter the bloodstream [35]. The production of LBP correlates with LPS levels, as more LBP is produced as LPS concentrations increase. We did not observe major differences in sCD14 levels across the entire study cohort, even though sCD14 is required as a co-factor for LBP in mediating innate immunity [36]. Previously, it was reported that sCD14 in mice has protective effects IBD, and an increase in the LBP/sCD14 ratio correlates with elevated plasma IL-6 levels [37], which is also observable in our study (Figure 2 ). Therefore, LBP, sCD14, and their ratio are considered crucial in regulating low-grade inflammation in inflammatory diseases. We did not observe differences in I-FABP levels (Figure 1 ), which are found in elevated levels in situations of epithelial damage [38] or increased levels of fecal inflammatory marker calprotectin, β-defensin-2 and serotonin (5-HT) between all test groups (Table 3 ). Interestingly, fecal zonulin family peptides, which increase permeability in the epithelium of the small intestine by modulating the intercellular tight junctions [39], yield in a lower signal in the PCS group, but the pairwise comparisons between individual groups were not significant. Thus, no signs of severe intestinal inflammation or epithelial damage could be observed. However, it’s important to note that intestinal barrier leakage occurs also in situations without an underlying damage or severe inflammation, as gastrointestinal mucosal tissues regulate their dynamics through intra- and inter-cellular transportation pathways [40]. Our findings regarding IL-1-β and IL-33 levels (Figure 2 ), which are related to gut microbiota homeostasis, support the hypothesis of mucosal barrier leakage [41, 42]. Further, we were able to show significant correlation between high LBP level and PCS.
However, our study has several limitations that should be considered. Due to the strict inclusion criteria, extensive medical records and laboratory work required, only a limited number of participants could be included. The number of ME/CFS patients is notably lower than in the other subgroups. Additionally, since the collection of anamnestic data relied mainly on open-ended questions, the self-reported data aspect provides an error risk.