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.