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viernes, 20 de octubre de 2023
Serotonin reduction in post-acute sequelae of viral infection
El COVID persistente reduce los niveles de serotonina circulante.
La deficiencia de serotonina afecta la cognición a través de una reducción de la señalización vagal.
Esto da una posible explicación a los síntomas neurocognitivos del COVID persistente.
Serotonin reduction in post-acute sequelae of viral infection
Post-acute sequelae of COVID-19 (PASC, “Long COVID”) pose a significant
global health challenge. The pathophysiology is unknown, and no
effective treatments have been found to date. Several hypotheses have
been formulated to explain the etiology of PASC, including viral
persistence, chronic inflammation, hypercoagulability, and autonomic
dysfunction. Here, we propose a mechanism that links all four hypotheses
in a single pathway and provides actionable insights for therapeutic
interventions. We find that PASC are associated with serotonin
reduction. Viral infection and type I interferon-driven inflammation
reduce serotonin through three mechanisms: diminished intestinal
absorption of the serotonin precursor tryptophan; platelet
hyperactivation and thrombocytopenia, which impacts serotonin storage;
and enhanced MAO-mediated serotonin turnover. Peripheral serotonin
reduction, in turn, impedes the activity of the vagus nerve and thereby
impairs hippocampal responses and memory. These findings provide a
possible explanation for neurocognitive symptoms associated with viral
persistence in Long COVID, which may extend to other post-viral
syndromes.
A major post-viral syndrome is “Long COVID,” manifesting as post-acute
sequelae of COVID-19 (PASC), which are experienced by a subset of
individuals after SARS-CoV-2 infection.
The molecular etiology of most post-viral syndromes, including Long
COVID, remains unclear. Several hypotheses have been proposed to explain
the persistence of symptoms, including the presence of a viral
reservoir that is not cleared after the initial infection,
A deeper understanding of whether these mechanisms occur in different
subsets of patients or jointly drive disease persistence is urgently
needed.
In this study, we perform a
metabolomics investigation and find that serotonin levels are a possible
discriminator between recovered individuals and Long COVID patients.
Using a combination of human cohort studies, animal models of viral
infection, and organoid cultures, we determine that the presence of
viral RNA and downstream interferon responses cause a decrease in
serotonin. Several mechanisms account for this phenomenon, including
diminished uptake of the serotonin precursor tryptophan in the
gastrointestinal tract, reduced storage in platelets due to
thrombocytopenia, and enhanced turnover by serotonin-metabolizing
enzymes. One important consequence of peripheral serotonin deficiency is
reduced activity of the vagus nerve, which in turn is associated with
hippocampal dysfunction and memory loss. Our findings suggest that many
of the current hypotheses for the pathophysiology of PASC might be
interconnected and offer actionable therapeutic insights.
Results
PASC can be characterized by serotonin reduction
We
began our explorations by defining a consensus metabolomics signature
of acute COVID-19. We integrated previously published metabolomics
datasets across different cohort studies
and ranked the metabolites detected in COVID-19 patients by their degree of deviation from the healthy state (Figures 1A and 1B ). Among the metabolites most strongly altered during acute COVID-19 were amino acids and their derivatives (Figure 1B).
We thus focused on possible roles for these metabolites in Long COVID.
We followed a cohort of 1,540 individuals with PASC at Penn Medicine and
performed a systematic symptom analysis based on questionnaire surveys
and chart review (Figures 1C and S1A–S1C; Table S1). Dimensionality reduction analysis defined eight subtypes of PASC based on symptom similarity (Figure 1D),
categorized by different degrees of initial hospitalization for acute
infection, mobility impairment, visceral malaise, cardiorespiratory
problems, and neurocognitive symptoms (Figures 1D–1F and S1D–S1S). We then performed targeted plasma metabolomics on 58 Long COVID patients who were representative of different clusters (Figure S1T) and experienced persistent symptoms 3 to 22 months after acute infection (Figure S2A). We compared them to 60 individuals with acute COVID-19 and 30 individuals with symptom-free recovery from COVID-19 (Figures 1G and S2B–S2D; Table S2).
Notably, the metabolite profile of Long COVID patients was distinct
from individuals who recovered to a symptom-free state after SARS-CoV-2
infection (Figure 1H).
To determine those molecules that drive the altered metabolomics state
in Long COVID, we correlated the abundance of each amino acid metabolite
with the presence of symptoms (Figure S2E). We identified a set of molecules whose levels were depleted in both acute and post-acute COVID-19 (Figure 1I), the most significant of which was serotonin (5-hydroxytryptamine, 5-HT) (Figures 1J and S2E).
In the post-acute state of infection, serotonin levels were predictive
of whether a patient fully recovered or developed long-term sequelae (Figure S2F).
Several other amino acids and their derivatives were either unaffected
during acute COVID-19 or returned to normal levels in both recovered
individuals and Long COVID patients (Figures S2G–S2I).
), serotonin was among the metabolites whose abundance was most strongly depleted in individuals with PASC (Figures S2J–S2L). In contrast, no serotonin reduction was observed in participants of the UNCOVR cohort
(Figures S2M and S2N; Table S3).
In this study, patients were enrolled during acute COVID-19 and then
longitudinally provided follow-up blood samples and symptom
questionnaires.
Conversely, the participants at Penn Medicine were enrolled after
seeking treatment at a post-COVID clinic. We thus speculated that the
severity of PASC might be greater in a cohort that presents for
treatment than in a longitudinal recovery cohort. Indeed, the average
number of symptoms was higher in the Penn Medicine cohort compared to
UNCOVR (Figure S2O).
To corroborate whether different levels of circulating serotonin can be
explained by differences in PASC symptoms, we measured plasma serotonin
levels in a separate longitudinal study (UCSF LIINC cohort
), which includes individuals with a wide range of symptoms (Figure S2P; Table S4).
Indeed, in this cohort, serotonin levels negatively correlated with the
number of symptoms that participants reported three to four months
after acute infection (Figure S2Q). Serotonin levels during the acute phase of SARS-CoV-2 infection were not predictive of the development of PASC (Figures S2R
and S2S). Taken together, these investigations reveal that serotonin
levels are diminished during acute COVID-19 and remain reduced in severe
cases of PASC.
we investigated the mechanisms underlying its decrease during acute
infection and Long COVID. We first explored whether serotonin depletion
was unique to COVID-19 or whether other acute viral infections led to a
similar decrease. To this end, we measured serotonin levels in the
plasma of 33 individuals with non-SARS-CoV-2 systemic viral infections
and compared them to 20 healthy controls (Figures 2A and S3A–S3D; Tables S5 and S6). As in acute COVID-19, serotonin levels were strongly decreased by other viral infections (Figure 2B), suggesting that this might be a more general characteristic of systemic viral infection.
To
investigate the mechanisms underlying this association, we used mouse
models of viral infection. We first infected mice expressing human ACE2
(K18-ACE2) with the ancestral strain of SARS-CoV-2 (Figures 2C and 2D). Notably, SARS-CoV-2 infection of K18-ACE2 mice led to a reduction in circulating serotonin (Figure 2E). We also observed reduced serotonin in wild-type mice infected with the beta variant of SARS-CoV-2
(Figures 2C,
2F, and 2G). Consistent with our human cohorts, this was not a unique
property of SARS-CoV-2, since infection of mice with vesicular
stomatitis virus (VSV) similarly decreased plasma serotonin levels (Figures 2C, 2H, and 2I).
Several studies indicate that viral persistence might be a characteristic feature of PASC.
We addressed this question using the lymphocytic choriomeningitis virus (LCMV) mouse model of persistent viral infection (Figure 2C).
While serotonin levels returned to baseline after clearance of an acute
infection (LCMV Armstrong), chronic viral infection sustained serotonin
reduction (LCMV clone 13) (Figures 2J, 2K, and S3E–S3H).
We thus speculated that reduced serotonin levels in Long COVID might be
a consequence of unresolved inflammation induced by viral products. To
test this, we recreated viral-induced inflammation in the absence of a
replicating pathogen by repeatedly injecting mice with the synthetic
double-stranded RNA polyinosinic:polycytidylic acid (poly(I:C)), which
mimics viral replication intermediates. Notably, poly(I:C) treatment was
sufficient to diminish serotonin levels (Figures 2L and S3I) both in total plasma and in isolated platelets, which are the major reservoir of circulating serotonin (Figures S3J and S3K). This effect was reversible since normal serotonin levels were restored within a week of poly(I:C) cessation (Figure 2M).
Both
viral infection and poly(I:C) treatment induce type I interferon (IFN)
signaling. Indeed, exposure to SARS-CoV-2, infection with VSV,
persistence of LCMV, or injection of poly(I:C) all strongly upregulated
the levels of interferon-stimulated genes (ISGs; Figures S3L–S3O). Importantly, sustained elevation of type I interferons has been observed in Long COVID patients.
We therefore asked whether the interferon response caused serotonin
reduction. Inhibiting interferon signaling through the interferon alpha
receptor (IFNAR) prevented poly(I:C)-induced serotonin reduction (Figure 2N).
Moreover, mice with genetic deficiency in either the poly(I:C) receptor
TLR3 or in the ISG-inducing transcription factor STAT1 (Figures S3P and S3Q) were resistant to the effects of poly(I:C) on serotonin levels (Figures 2O
and 2P). Serotonin depletion did not appear to contribute to host
defense, since pharmacological inhibition of the serotonin-synthesizing
enzyme TPH1 enhanced viral loads and pathogenesis during VSV infection
and had no effect on SARS-CoV-2 replication (Figures S3R–S3V).
Collectively, these findings suggest that the canonical pathway of
viral RNA sensing and type I interferon induction by TLR3 leads to
serotonin depletion (Figure 2Q).
We
next investigated the mechanisms by which viral-induced inflammation
reduces serotonin levels. The large majority of circulating serotonin is
produced in the gastrointestinal tract, where it is synthesized from
dietary tryptophan in enterochromaffin cells
(Figure 3A).
We thus investigated whether serotonin production during viral
infection might be limited by reduced tryptophan availability. Indeed,
individuals with acute COVID-19 showed reduced plasma tryptophan levels (Figures 1B and 3B).
Moreover, tryptophan levels were decreased in Long COVID patients (Figures 1B and 3B).
A similar decrease in tryptophan levels was observed in the UCSF LIINC
cohort and in another independent Long COVID study (Rush University)
(Figures S4A and S4B; Table S4). Plasma tryptophan concentrations were likewise reduced during chronic LCMV infection and after poly(I:C) treatment of mice (Figures 3C
and 3D), suggesting that lower tryptophan availability may cause
serotonin reduction by substrate limitation. Consistently, feeding a
tryptophan-deficient diet to mice phenocopied the effect of poly(I:C)
treatment on plasma serotonin levels in mice (Figures 3E and 3F).
Generally,
tryptophan deficiency can be caused by either reduced intestinal
absorption or by enhanced conversion into tryptophan derivatives such as
kynurenine (Figure 3A).
Kynurenine levels are elevated during viral infection, and numerous
reports have highlighted kynurenine as a metabolite strongly induced by
SARS-CoV-2 infection
(Figure 1B). Indeed, kynurenine levels were increased during acute COVID-19 in our cohort (Figure S4C) and likewise elevated by poly(I:C) treatment of mice (Figure S4D).
We therefore hypothesized that serotonin reduction was a consequence of
tryptophan depletion due to increased kynurenine production. However,
the increase in kynurenine levels did not persist in individuals with
PASC (Figure S4C).
Furthermore, mice lacking the kynurenine-producing enzyme IDO1, which
are deficient in kynurenine production, still presented with reduced
serotonin upon poly(I:C) treatment (Figures S4E
and S4F). Similarly, pharmacological inhibition of the alternative
kynurenine-producing enzyme TDO2 did not restore serotonin levels (Figures S4G
and S4H). These findings make it unlikely that kynurenine production is
the major cause for serotonin depletion during viral inflammation.
We
therefore explored intestinal amino acid uptake as a possible cause of
tryptophan deficiency and serotonin depletion. Since poly(I:C) treatment
reduces food intake (Figure S4I),
we speculated that tryptophan deficiency may result from diminished
consumption of this essential amino acid. However, the poly(I:C)-induced
tryptophan and serotonin reduction was seen even after an extended
fast, in paired feeding experiments, and in experiments in which we
supplemented food to poly(I:C)-injected mice (Figures S4J–S4N).
The number of serotonin-producing enterochromaffin cells was unaltered
by poly(I:C) treatment, ruling out enzymatic synthesis of serotonin as
the critical bottleneck (Figures S4O
and S4P). We thus used an unbiased approach to explore the impact of
viral inflammation on intestinal nutrient absorption. We performed
RNA-sequencing of small intestinal tissue of poly(I:C)-treated mice and
controls, which revealed strong alterations in intestinal gene
expression (Figure S4Q). Expectedly, most upregulated genes belonged to viral recognition and inflammation pathways (Figures 3G and S4R).
Remarkably, the gene functions most significantly diminished by
poly(I:C) treatment were involved in nutrient metabolism, including
amino acid absorption (Figures 3G–3I, S4R, and S4S). For example, the expression of the apical global amino acid transporter ATB0,+ (Slc6a14), the neutral amino acid transporter B0AT1 (Slc6a19), and the B0AT1 chaperone ACE2 were all strongly decreased in poly(I:C)-treated mice (Figures 3G and 3J). The expression of transporters on the basolateral side, such as LAT2 (Slc7a8), were likewise reduced (Figures 3G
and 3J). In contrast, the biosynthetic pathway converting tryptophan
into serotonin, including the rate-limiting enzyme TPH1, was not
affected (Figure 3J).
These data highlight transcriptional downregulation of key amino acid
absorption genes during viral inflammation, which we verified by qPCR of
intestinal tissue from poly(I:C)-treated mice (Figures S5A–S5K).
Figure S5The transcriptional impact of viral inflammation, related to Figure 4
We
next used both mice and intestinal organoids to reconstruct the
poly(I:C)-induced signaling pathway leading to transcriptional
alteration in tryptophan uptake genes (Figures S5A and S5L). As in intestinal tissue, small intestinal organoids responded to poly(I:C) with downregulation of Ace2 and Slc6a19 (Figures 4A , 4B, and S5M). TLR3 deletion prevented the downregulation of these genes after poly(I:C) injection (Figures 4C
and 4D). Inhibition of the transcription factor NF-κB, which signals
downstream to TLR3, blunted the induction of an interferon response and
the downregulation of Ace2 and Slc6a19 in organoids (Figures 4E, 4F, S5N,
and S5O). Notably, exposure to type I interferons was sufficient to
reduce the expression of genes involved in tryptophan absorption (Figures 4G, 4H, and S5P).
The interferon receptor signals via STAT1, and we verified marked STAT1
phosphorylation in response to poly(I:C) treatment in both organoids
and intestinal epithelial cells (Figures S5Q and S5R). STAT1 was required for the transcriptional inhibition of Ace2 and Slc6a19 (Figures 4I and 4J) in an epithelial-intrinsic manner (Figures 4K and 4L).
Figure 4Mechanisms of viral inflammation-induced intestinal gene expression changes
To
explore the connection between viral persistence in the gut and
transcriptional regulation of tryptophan uptake genes, we examined
gastrointestinal samples from both mice and humans after viral
infection. Indeed, we observed downregulation of Ace2 and Slc6a19 in both acute (VSV) and chronic (LCMV clone 13) settings of viral infection (Figures 4M–4P). Acute SARS-CoV-2 infection in mice also resulted in detectable viral RNA in intestinal tissue (Figures 4Q and 4R), and data from SARS-CoV-2-infected human intestinal organoids
revealed strong transcriptional inhibition of ACE2 and SLC6A19 (Figures 4S
and 4T). Numerous reports have suggested that SARS-CoV-2 can replicate
in the human gastrointestinal tract and remain detected long after the
acute infection.
We confirmed these findings in tissue samples obtained from autopsies
during the acute (<2 weeks) and post-acute (>2 weeks) phase after
SARS-CoV-2 infection (Figure 4U). While viral RNA could be amplified from several organs during the acute phase (Figure 4V), the gastrointestinal tract stayed viral-RNA-positive in samples obtained from the post-acute phase (Figures 4V and S5S).
To determine whether viral persistence in the gastrointestinal tract
was associated with the development of PASC, we collected stool samples
from individuals with PASC as well as a control group of individuals
with prior SARS-CoV-2 infection but no persistent symptoms (Figure 4U). Viral RNA was indeed detected in the stool of a subset of individuals with PASC (Figure 4W),
highlighting a possible connection between the presence of viral
components in the gastrointestinal tract and the persistence of
long-term symptoms in certain individuals.
We
next assessed the consequences of reduced epithelial expression of
amino acid uptake genes during viral inflammation. In addition to
tryptophan, we noted a pronounced reduction in the plasma concentrations
of several amino acids in mice injected with poly(I:C), particularly in
neutral amino acids (Figure 5A). This amino acid profile resembled the one in mice lacking ACE2 (Figure 5B), which together with B0AT1 is required for the transport of neutral amino acids across the apical membrane of intestinal epithelial cells.
We confirmed that the successive loss of functional Ace2 alleles in heterozygous and homozygous Ace2-deficient mice led to a stepwise reduction in tryptophan levels (Figure 5C). Mice lacking ACE2 were also unable to absorb an oral bolus of tryptophan (Figure 5D), in line with previous findings.
Notably, the same phenomenon was observed with poly(I:C) treatment of heterozygous Ace2-deficient mice (Figure 5E), indicating that transcriptional downregulation of Ace2 in these mice phenocopied the homozygous Ace2-deficient state. While the systemic levels of tryptophan were reduced, ileal tryptophan accumulated after poly(I:C) injection (Figures 5F and 5G). Isotope tracing confirmed that circulating tryptophan is derived from the orally supplemented source (Figure S6A), highlighting that poly(I:C) treatment prevented tryptophan absorption.
If
tryptophan uptake was abrogated by poly(I:C) treatment, tryptophan
supplementation should elevate serotonin levels even during viral
inflammation. To corroborate this, we used a diet containing
a glycine-tryptophan dipeptide, which bypasses the need for B0AT1 and enables tryptophan uptake via dipeptide transporters.
This diet compensated for impaired uptake in poly(I:C)-treated mice and
led to an increase in both tryptophan and serotonin levels in systemic
circulation (Figures 5H
and 5I). Similarly, supplementation with the serotonin precursor
5-hydroxytryptophan (5-HTP), which bypasses the requirement for
tryptophan, rescued serotonin levels in poly(I:C)-injected mice (Figure 5J).
Collectively, these data demonstrate that viral-RNA-induced
inflammation impairs intestinal tryptophan uptake, which causes systemic
serotonin depletion (Figure 5K).
Viral inflammation impairs serotonin storage
Upon
synthesis in enterochromaffin cells, circulating serotonin is
transported inside platelets, while free serotonin is rapidly degraded
by monoamine oxidase (MAO) enzymes (Figure 6A).
providing a possible explanation for reduced circulating serotonin levels (Figures 6B–6D). Poly(I:C)-induced thrombocytopenia was dependent on the TLR3-IFN-STAT1 signaling pathway (Figures 6E–6G). The overall white blood cell count was unchanged by poly(I:C) treatment (Figure S6B). Erythrocyte, hemoglobin, and hematocrit counts were reduced (Figures S6C–S6E), while mean corpuscular volume and mean corpuscular hemoglobin were not affected (Figures S6F and S6G). Increased mean platelet volumes (Figures 6H and 6I) were indicative of increased destruction of platelets,
which was likewise dependent on TLR3, type I interferon signaling, and STAT1 (Figures 6J–6L). Tryptophan supplementation was unable to restore platelet counts (Figure S6H),
indicating that reduced intestinal amino acid uptake and platelet
depletion were independent effects of poly(I:C) injection. Consistently,
platelet depletion
We
next investigated the causes for thrombocytopenia during viral
inflammation. The number and size of megakaryocytes in the bone marrow
was increased in poly(I:C)-treated mice (Figures S6L–S6N), while thrombopoietin levels were unchanged (Figures S6O and S6P). We noted that the baseline activation status of platelets was increased by poly(I:C) treatment (Figures 6N and 6O). Consistently, platelet aggregation was markedly enhanced (Figures 6P and 6Q). Prothrombin time (PT) and partial thromboplastin time (PTT) were reduced (Figures 6R and 6S), further indicative of hypercoagulability.
We ruled out changes in the concentrations of fibrinogen, tissue factor, or TAT complexes as alternative explanations (Figures S6Q–S6S).
Collectively, these results indicate that viral inflammation drives
platelet hyperactivation, resulting in hypercoagulability and
thrombocytopenia in an interferon-dependent manner. Consequently,
platelet-mediated systemic serotonin transport is impaired.
Since free serotonin is the target of rapid degradation,
we next focused on MAO-mediated serotonin turnover. We found that intestinal transcript levels of Maoa were increased in SARS-CoV-2-infected, VSV-infected, and poly(I:C)-treated mice in a TLR3-dependent manner (Figures 6T–6V and S6T).
Consistently, the levels of the serotonin degradation product
5-hydroxyindoleacetic acid (5-HIAA) were increased in the urine of
virally infected mice and in mice injected with poly(I:C) (Figures 6W–6Y and S6U). STAT1-deficient mice were protected from the accumulation of 5-HIAA (Figure 6Z).
Notably, pharmacological inhibition of MAO prevented the accumulation
of 5-HIAA and restored serotonin levels in poly(I:C)-treated mice (Figures 6AA and S6V). These findings indicate that serotonin turnover is enhanced during viral inflammation.
Serotonin reduction impairs vagal signaling and memory function
Finally,
we explored the consequences of peripheral serotonin depletion on
individuals experiencing PASC. In a symptom questionnaire administered
at the time of blood draw, the majority of patients in our cohort
reported fatigue, cognitive difficulties, headaches, loss of endurance,
problems with sleep, anxiety, and memory loss (Figure S7A).
To investigate possible mechanisms underlying the association between
serotonin reduction and prevalent neurocognitive manifestations, we
again turned to mouse models. We observed cognitive impairment in the
setting of acute VSV infection, chronic LCMV persistence, and in
poly(I:C)-treated mice as assessed by the novel object recognition
paradigm
(Figures 7D and 7E). Platelet depletion similarly impaired memory function (Figure 7F).
We therefore hypothesized that serotonin reduction may be responsible
for poor cognitive performance after poly(I:C) injection. Indeed,
treatment of mice with the selective serotonin reuptake inhibitor (SSRI)
fluoxetine restored novel object recognition (Figure 7G),
and rescue of tryptophan levels by glycine-tryptophan supplementation
reinstated normal cognitive performance in poly(I:C)-treated mice (Figure 7H). Differences in explorative behavior did not affect the results across all of these experiments (Figures S7B–S7H).
Figure S7Cognitive performance during viral inflammation, related to Figure 7
We found that hippocampal activation in response to novel object exposure was blunted in poly(I:C)-treated mice (Figures 7I–7K, S7I, and S7J). This was not accounted for by changes in hippocampal neurogenesis (Figures S7K–S7N). Since serotonin plays an important role in hippocampal function,
we hypothesized that serotonin reduction directly impaired the
generation of hippocampus-dependent memories. However, serotonin levels
in the brain were unaffected by viral inflammation (Figure 7L), suggesting that the peripheral reduction of serotonin was responsible for cognitive impairment.
Circulating serotonin does not cross the blood-brain barrier
To explore the impact of peripheral serotonin on sensory neurons, we
measured neuronal activation in sensory terminals of the nucleus tractus
solitarii (NTS) in the brainstem. Novelty exposure led to an increase
in cFos+ cells in the NTS, but this response was abrogated upon poly(I:C) treatment (Figures 7M
and 7N), suggesting that serotonin depletion causes cognitive
impairment through reduced sensory neuron activity. Consistently,
restoration of peripheral serotonin levels using 5-HTP rescued cognition
in poly(I:C)-treated mice (Figures 7O and S7O), and so did the TRPV1 agonist capsaicin, a strong stimulant of sensory neurons (Figure 7O). Of note, capsaicin treatment did not affect peripheral serotonin levels (Figure S7O), and neither capsaicin nor 5-HTP treatment ameliorated poly(I:C)-induced ISG responses in the brain (Figure S7P),
highlighting that restoration of sensory input from the periphery is
able to rescue cognition despite serotonin deficiency or ongoing
neuroinflammation. Peripheral serotonin reduction alone, as in the case
of platelet depletion, did not trigger inflammation in the brain (Figure S7Q).
TRPV1+
sensory neurons can be broadly categorized as vagal and spinal cord
afferents. To distinguish between both possibilities, we
chemogenetically activated Phox2b-expressing neurons, which are
restricted to the vagus nerve. Indeed, stimulation of Phox2b neurons
during poly(I:C) treatment restored activation of hippocampal neurons
and the formation of short-term memories (Figures 7P–7R and S7R). To determine the mechanism by which serotonin influences the activity of vagal neurons, we used an in vitro
system in which we cultured neurons from nodose ganglia and exposed
them to serotonin. Vagal neurons robustly responded to serotonin
treatment, as evidenced by rapid calcium influx (Figure 7S), suggesting a possible direct effect of peripheral serotonin on the vagus nerve. Single-cell transcriptomics data
showed high and selective expression of the serotonin receptor 5-HT3 on vagal neurons (Figure S7S). To determine whether serotonin signaling via 5-HT3 receptors was sufficient to restore cognition during viral inflammation, we used the pharmacological 5-HT3 receptor agonist meta-Chlorophenylbiguanide (m-CPBG). Indeed, m-CPBG
treatment normalized both novelty responses of hippocampal neurons and
performance in the novel object recognition paradigm (Figures 7T
and 7U). Taken together, these findings suggest that serotonin
reduction dampens vagal signaling and thereby impairs cognitive
function.
Discussion
The emergence of PASC poses a global health challenge. The pathophysiology of post-viral syndromes remains poorly understood,
leaving medical systems across the world unprepared for the large
number of individuals developing cardiorespiratory, neurocognitive,
gastrointestinal, and musculoskeletal symptoms in the months and years
A deeper understanding of the molecular and cellular etiopathology of PASC is thus urgently needed.
In
this study, we have investigated metabolite signatures associated with
Long COVID. We have focused on metabolites whose concentrations are
perturbed both in acute COVID-19 and in patients with PASC. Among the
metabolites we measured, the molecule most significantly associated with
PASC was serotonin. We show that viral inflammation-driven serotonin
depletion can be caused by reduction of tryptophan absorption,
thrombocytopenia, and increased MAO expression. This response is TLR3-,
IFNAR-, and STAT1-dependent and results in decreased vagal and
hippocampal activation as well as cognitive impairment.
These
findings have several important implications. First, they highlight the
profound consequences that persistent viral reservoirs can have.
Numerous studies have provided evidence for the presence of viral
components
Our data indicate that the presence of viral components and resultant
interferon response might be a causative factor in the development of
PASC-associated symptoms.
Second,
our study highlights a mechanism by which viral infection can alter
amino acid uptake. Deviations from homeostatic concentrations of amino
acids can exert profound effects on tissue function.
While we focused on serotonin in this study, tryptophan serves as the
precursor for many other important metabolites, including niacin, NAD,
and melatonin.
The evolutionary teleology of reduced intestinal amino acid absorption
during viral inflammation remains unclear, but it is possible that acute
downregulation of genes involved in amino acid uptake is part of a
cellular response to interferon stimulation aimed at abrupt cessation of
cellular metabolism during viral infection. In the case of
non-resolving viral inflammation, this response may persist and result
in nutrient deficiency.
Third, a
common feature of both acute and post-acute SARS-CoV-2 infection is the
formation of microthrombi as a result of hypercoagulability.
Our findings imply that thrombocytopenia may diminish the carrying
capacity of the systemic circulation for serotonin. Reduced serotonin
storage, coupled with the induction of MAO enzymes, may enhance the
turnover of serotonin and excretion of its degradation products. Thus,
hypercoagulability in acute COVID-19 and Long COVID may have
implications beyond its cardiovascular effects.
Fourth, our study indicates a role for the vagus nerve in mediating the impact of serotonin reduction on the brain.
Since unequivocal evidence for SARS-CoV-2 replication in the brain is
lacking, recent studies have focused on the cognitive consequences of
peripheral immune activation as well as neuroinflammation.
Based on our data, we suggest that afferent sensory neurons may play a
critical role in the neurocognitive manifestations of both acute and
post-acute viral infections. The vagus nerve is an important mediator of
sickness behavior,
While the precise circuit by which the vagus nerve is involved in the
development of PASC remains unclear, sensory neurons may emerge as an
important element in relaying the effect of peripheral viral
inflammation to the brain.
Finally,
our findings indicate possible targets for clinical interventions aimed
at the prevention and treatment of PASC. Our animal models demonstrate
that serotonin levels can be restored and memory impairment reversed by
precursor supplementation or SSRI treatment. While the effectiveness of
SSRIs in acute COVID-19 has been a subject of debate,
no systematic exploration of SSRIs in individuals with PASC has been
performed to date. Our study, together with recent findings linking
depression with cognitive impairment in Long COVID
it is possible that virus-induced receptor internalization augments the
effect of interferons on ACE2 downregulation and serotonin reduction.
In principle, however, none of the mechanisms described in this study
are unique to SARS-CoV-2 infection. Indeed, reduced serotonin levels
have been reported in other settings of viral inflammation, such as
dengue virus infection,
The connection between serotonin reduction and vagus nerve dysfunction
may thus be relevant beyond Long COVID. The fact that low serotonin
levels are also found in non-viral conditions characterized by elevated
interferon levels, such as systemic lupus erythematosus or multiple
sclerosis,
suggests that the pathway described in this study may even apply beyond viral infections.
Limitations of this study
The
degree of serotonin reduction is variable across the four cohorts of
individuals with PASC that we have examined in this study. While modes
of recruitment, number of symptoms, and degree of disease severity might
provide possible sources of this variability, there are likely further
differences that we have not accounted for. The manifestations of Long
COVID are highly heterogeneous,
and the subtypes of PASC that are studied in individual cohorts are
likely different. Our results indicate that serotonin reduction is not
specific to any particular subset of PASC, but much larger numbers of
longitudinal samples are required to comprehensively characterize serum
metabolite levels across the different endotypes of Long COVID.
In
addition, while we provide evidence for serotonin reduction in acute
COVID-19, individuals with PASC, and acutely and chronically infected
mice, mouse models for Long COVID are still lacking, and thus our study
does not establish a direct causal connection between post-acute
SARS-CoV-2 infection, tryptophan uptake, thrombocytopenia, and serotonin
levels. The chronic LCMV and poly(I:C) models used in this study
recapitulate important features of SARS-CoV-2 infection but have clear
limitations. For instance, when administered systemically, poly(I:C) may
not accurately mimic the tissue-level inflammatory processes induced by
persistent viral reservoirs. Furthermore, while the persistent presence
of circulating spike protein may be a useful marker for PASC,
it remains unclear whether remnants of SARS-CoV-2 nucleic acid play any functional role in Long COVID.
Finally,
our assessment of viral persistence in the gastrointestinal tract of
individuals with PASC is based on a limited number of participants.
Similarly, we have not demonstrated a direct connection between
intestinal viral persistence and chronically elevated levels of type I
interferons in humans, which would require collecting a large number of
intestinal biopsies from Long COVID patients. Our results thus call for
the large-scale investigation of the causal connection between the
presence of a viral reservoir in the gastrointestinal tract, sustained
inflammatory responses, and manifestations of Long COVID.
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