Gut-brain communication in COVID-19: molecular mechanisms, mediators, biomarkers, and therapeutics

Abstract

Introduction:

Infection with COVID-19 results in acute respiratory symptoms followed by long COVID multi-organ effects with time presenting with neurological, cardiovascular, musculoskeletal, and gastrointestinal (GI) manifestations. Temporal relationship between gastrointestinal and neurological symptoms is unclear but is warranted for exploring better clinical care for COVID-19 patients.

Areas covered:

We critically reviewed the temporal relationship between gut-brain axis after SARS-CoV-2 infection and the molecular mechanisms involved in neuroinvasion following GI infection. Mediators are identified that could serve as biomarkers and therapeutic targets in SARS-CoV-2. We discussed the potential therapeutic approaches to mitigate the effects of GI infection with SARS-CoV-2.

Expert opinion:

Altered gut microbiota cause increased expression of various mediators, including zonulin causing disruption of tight junction. This stimulates enteric nervous system and signals to CNS precipitating neurological sequalae. Published reports suggest potential role of cytokines, immune cells, BOAT1 (SLC6A19), ACE2, TMRSS2, TMPRSS4, IFN-γ, IL-17A, zonulin, and altered gut microbiome in gut-brain axis and associated neurological sequalae. Targeting these mediators and gut microbiome to improve immunity will be of therapeutic significance. In-depth research and well-designed large-scale population-based clinical trials with multidisciplinary and collaborative approaches are warranted. Investigating the temporal relationship between organs involved in long-term sequalae is critical due to evolving variants of SARS-CoV-2.

1. Introduction

Infection with SARS-CoV-2 has resulted in an ongoing global pandemic and widespread vaccination efforts. The most common symptoms of SARS-CoV-2 are fever, cough, fatigue, and dyspnea at 81.2%, 58.5%, 38.5%, and 26.1%, respectively [1] along with others including gastrointestinal, neurological, respiratory, musculoskeletal, and cardiovascular manifestations [2, 3]. . As the number of SARS-CoV-2 cases rises, there is more supporting data of neurological manifestation of SARS-CoV-2. One study from Wuhan, China found that 36.4% of 214 COVID-19 infected individuals had neurological symptoms. Neurological symptoms were most prevalent in severe-COVID-19 cases. The most common neurological symptoms included headache and dizziness at 17% and 13%, respectively [4]. There is also evidence of long-lasting neurological effects from SARS-CoV-2 complications after recovery. These long-term effects include cerebrovascular disease, impaired consciousness, and muscular injury [2, 4, 5]. Multiple experimental models have used human coronavirus to explore possible autoimmune changes seen in multiple sclerosis. The findings are reported that coronavirus is recognized by Toll-like receptors (TLRs) and contribute to the immune response in multiple sclerosis. Such TLRs are involved in the pathogenesis of multiple sclerosis. This interaction shows a potential communication between coronavirus and the demyelinating diseases. Investigating the temporal relationship between SARS-CoV-2 and multiple sclerosis is important because infection with SARS-CoV-2 can exacerbate the symptoms of multiple sclerosis possibly by neuroinvasion, neuroinflammation, and stimulation of autoreactive T cells against myelin [6-8]. These complications could lead to lethal outcomes even after recovering from the infection. Encephalitis, demyelination, neuropathy, and stroke associated with SARS-CoV-2 indicate the extent and specific involvement of the nervous system [2, 9]. Apart from central nervous system (CNS), peripheral nervous system manifestations including nerve pain, skeletal muscle injury, cranial polyneuritis, neuro-ophthalmological disorders, Guillain-Barré syndrome, neuromuscular junction disorders, neurosensory hearing loss, and dysautonomia associated with SARS-CoV-2 have also been reported [10].

In addition to neurological manifestations, there are reports of gastrointestinal (GI) symptoms associated with SARS-CoV-2. Lin et al. [11] reported that the prevalence of diarrhea, anorexia, and nausea were 24.2%, 17.9%, and 17.9%, respectively in hospitalized patients with SARS-CoV-2. Moreover, 40% of patients with severe acute respiratory syndrome have associated diarrhea and other intestinal problems. Patients with diarrhea have commonly required respiratory assistance or admission to the intensive care unit [12]. Given this association, the severity of GI symptoms can be used as an indicator for the severity of SARS-CoV-2.

Interestingly, there exists a correlation between the frequency of neurological and GI symptoms of COVID-19. One epidemiological study analyzing 945 patients found that the neurological and GI symptoms were 54.5% and 53.2%, respectively. Those with GI symptoms also significantly (p=0.027) presented with neurological symptoms [13]. However, the underlying mechanism of this correlation remains unclear. The RNA of SARS-CoV-2 has been detected in the CNS and stool of COVID-19 infected individuals [14, 15]. The association could be an important prognostic factor before severe neurological symptoms such as cerebral vascular events occur.

A study published recently by Ebrahim et. al. [16], investigated the relationships between acute GI symptoms, COVID-19 severity, and post-COVID-19 gut-brain interaction. The study found that GI symptoms were independent risk factors for the severity of COVID-19 and that 66% of individuals with SARS-CoV-2 and GI symptoms had features of post-COVID-19 Disorders of Gut-Brain Interaction (DGBI). The diagnosis of DGBI here was made by analyzing a Rome IV Adult Questionnaire [16]. In a recent study of meta-transcriptome sequencing of bronchoalveolar lavage fluid, gut-lung axis was reported with the findings that the microbiota of SARS-CoV-2 patients contained many pathogens from oral and upper respiratory commensal bacteria. There are few reports about gut-brain and gut-lung axis but most of the research has focused on the respiratory impact of infected individuals with SARS-CoV-2 and the literature investigating the temporal relation between gastrointestinal, neurological, respiratory, musculoskeletal, and cardiovascular sequalae of SARS-CoV-2 infection is limited. A gut-brain axis has been established and data support the hypothesis that the gut may be an entering port by which SARS-CoV-2 viruses enter and ultimately various cytokines and neurotoxins migrate to the brain causing damage to the nervous system. For instance, a study by Troisi et al [17] describes that both the respiratory and GI tracts have the highest expression of angiotensin-converting enzyme-2 (ACE2) receptors. Tissues with the highest expression of ACE2 have a high sensitivity to infection. In this article, we critically summarized and discussed these pathways and biomarkers through which the GI system could influence neurologic sequelae in COVID-19.

1.1. Long-COVID and gut-brain axis

Initially, concerns about SARS-CoV-2 were primarily related to the effects of acute infection stages. As time elapsed and more people became infected and recovered from SARS-CoV-2, there have been increased reports of long-term effects of COVID-19 that persisted for many months after the initial infection called Long COVID. Many studies have shown neurological and GI associations of Long COVID. In a meta-analysis with 11,324 post-COVID-19 infected patients stratified into hospitalized and outpatient groups, both groups showed symptoms such as fatigue, brain fog, memory issues, attention disorder, anosmia, dysgeusia, and headaches that lasted ≥3 months after initial infection [18]. Furthermore, this analysis demonstrated an increase in neuropsychiatric symptoms such as sleep disturbances, anxiety, and depression in post-COVID infection. Another study showed that 29% of patients had GI-related symptoms post-COVID infection such as diarrhea, constipation, nausea, vomiting, and abdominal pain [19]. The same set of patients reported that 37% had mental health symptoms after infection such as anxiety and sadness, possibly linking the GI symptoms to neuropsychiatric effects of COVID-19. The pathological mechanisms of Long COVID is not too well understood, however many articles support that it is related to the neuroinvasion of SARS-CoV-2 in the initial infection through the ACE2 receptor [20], as detailed in other sections of this article. The initial infection may have led to inflammation and oxidative stress in the brain tissue affecting areas of the brain such as the hippocampus, cortical atrophy, and ischemic vascular changes [21-23]. The brain of a group of 785 post-COVID participants was analyzed through magnetic resonance imaging in the United Kingdom and showed a reduction of the gray matter in the orbitofrontal cortex and parahippocampal gyrus, reduction of global brain size, and markers of tissue damage to the olfactory cortex [20]. Given this association in Long COVID, it is critical to understand the gut-brain association of SARS-CoV-2 and its effect on long-term effects on the GI and neurological systems.

2. Search strategy

A systematic search of PubMed and the Web of Science was performed between January 2019 through June 2022 using keywords: SARS-CoV-2 with gut-brain axis (34 results), enteric nervous system (19 results), gastrointestinal inflammation and gut microbiota (101 results), gastrointestinal inflammation and neuroinflammation (7 results), neuroinvasion (280 results), probiotics (84 results) and SARS-CoV-2 with neuroinflammation and sequalae (682 results), and long COVID with neurological sequalae (274 results). Article titles and abstracts were screened, with full-text review ultimately determining inclusion status. Full length original and review articles in English language discussing the temporal relationship between gastrointestinal inflammation and neurological symptoms in SARS-CoV-2 were included. Articles with only abstract or in language other than English were not included for this narrative review (Figure 1).

Figure 1:

Flow Chart of the Literature Search.

3. Association between gastrointestinal and neurological systems

The gut-brain axis is a heavily interconnected and complex system involving many neuropeptides, inflammatory markers, microbiota, and hormones [24]. The knowledge of the complex interplay between the gastrointestinal system and neurological system may shed novel insights into the neurological manifestations of COVID-19. Communication pathways of the gut-brain axis have not been well established, but proposed mechanisms involve the change in gut microbiome, inflammatory mediators, and enteric nervous system [24].

3.1. Altered gut microbiota

The gut microbiota is composed of thousands of microbial species, approximately 3.8x10¹³, outnumbering human cells [25]. The gut microbiome influences the immune system and subsequently the nervous system through the stimulation of local and systemic immune responses. The neurologic and psychological functions have a profound relationship with the gut, hence an increase in the number of studies investigating the gut-brain axis [26]. The microbiota influence CNS function through synthesizing neurotransmitters and mediators such as 5-hydroxytryptamine (5-HT [serotonin]), histamine, melatonin, acetylcholine, and catecholamines [27]. In COVID-19, the imbalance in the gut microbiota and a weakened immune system have systemic effects. Patients with gut symptoms present with increased IL-6 and fecal calprotectin, indicators of gut inflammation, and disrupted gut integrity [28]. Elevated IL-6 has been associated with SARS-CoV-2 RNA in critically ill patients [29]. Theories have suggested that COVID-19 patients may have a disrupted gut barrier that allows SARS-CoV-2 to enter the bloodstream and access the brain causing neurologic sequela [30]. Disruption of the gut barrier and altered microbiota are the probable mechanisms underlying neuroinvasion through GI system.

The hypothesis that SARS-CoV-2 entry altering gut microbiota may have neurological manifestation is supported by the fact that the gut microbiota influence not only immune response in the brain but also neurogenesis and cognitive function [31, 32]. Several findings suggest that impairment of gut microbiota in SARS-CoV-2 may have temporal association with neurological manifestations. These include the involvement of the gut microbiota in shaping gut-associated lymphatic tissue (GALT) and differentiation of interleukin-17 (IL-17)-associated Th17 cells [33, 34], GALT-induced CNS autoimmunity by activating CNS T-cells and auto-antibody-producing B cells [31], maturation and function of the microglia [35], enhanced impairments in Parkinson’s disease [36], and autism. Brundin et al postulate that systemic inflammation caused by severe COVID-19 can trigger neuroinflammation and nigral dopamine neurons. Dopamine neurons in the nigrostriatal pathway are affected by systemic inflammation, which in COVID-19 is highly linked to IL-6 levels and kyneurenine pathways [29, 37], the pathways associated with neurological disease [38, 39]. This complex interplay between the immune system, gut microbiome, and nervous system has been seen in numerous other neuropsychiatric and neuroinflammatory diseases and sheds light on the possible mechanisms of COVID-19 neuroinvasion [40, 41]. In summary, the gut microbiome plays an important role in influencing the nervous system and has been implicated in many neurologic disorders.

3.2. Inflammation

Gastrointestinal symptoms such as fever, myalgia, lethargy, dry cough, dyspnea, anorexia, abdominal pain, and diarrhea are common in SARS-CoV-2 patients and a significant association of these symptoms with neurological symptoms supports the notion of the altered GI tract during COVID-19 with neurological symptoms [13]. The presence of receptors and co-receptors for SARS-CoV-2 in enterocytes and on enteric mucosae of GI tract such as luminal sodium-dependent neutral amino acid transporter of the small intestine B(0)AT1 (SLC6A19), ACE2, and two isoforms of the serine protease TMRSS2 and TMPRSS4 and the presence of pH, clathrin- and caveolin1-dependent endocytosis occurring in cholesterol/sphingolipid-rich membrane lipid domains facilitating the entry of SARS-CoV-2 to the submucosal bloodstream through hepatic artery suggest the role of GI tract-mediated SARS-CoV-2 infection [42]. Once entered the bloodstream, SARS-CoV-2 affects the brain by entering through an altered blood-brain barrier. SARS-CoV-2-GI tract infection inducing cytopathic impacts through ACE2, and immune-mediated inflammatory cytokine storm and intestinal damage caused by drugs further aggravate the symptoms and can affect the brain through secreted cytokines [43]. The GI system has many direct and indirect effects on the immune system. One study shows that in mice, a diet high in salts suppresses cerebral blood flow leading to cognitive impairment [44]. This cognitive impairment can be traced to an inflammatory pathway in the gut, governed by Th17 cells and increased IL-17. IL-17, promoted by nitric oxide, here leads to endothelial dysfunction and ultimately cognitive impairment. Moreover, studies reveal that T cells travel from the gut to the brain, localize in the leptomeninges, and cause neuroinflammation by secreting IL-17 [45]. Increased IL-17 in the CNS then leads to chemokine production in the brain parenchyma, with a subsequent increase in other cytotoxic immune cells. Theories suggest that T cells enter the nervous system through the choroid plexus into the cerebrospinal fluid and drain through the meningeal lymphatic system to the deep cervical lymph nodes [46]. Others suggest that intestinal T cells directly accumulate in the meninges as opposed to trafficking through the cervical lymph nodes first, revealing a direct gut-brain communication.

Additionally, the gut mediates the pathogenesis of neuroinflammatory diseases, such as multiple sclerosis, through IFN-γ, IL-17A, and zonulin [47]. IFN-γ, IL-17A, and zonulin increase the permeability of the small intestinal epithelial barrier (IEB), and in vitro studies reveal that they increase the permeability of blood-brain barrier by modifying tight junctions (TJ) and the actin cytoskeleton. An increase in IL-17 is implicated in endothelial dysfunction, cognitive impairment, and the permeability of the blood-brain barrier. There also exists a complex interplay between Th17, IL-17A, IFN-γ, and zonulin in the gut-brain axis in COVID-19. This relationship is discussed in more depth in the following sections.

3.3. Enteric nervous system

The enteric nervous system (ENS), often referred to as the “second brain” is an extensive system for digestion that works with the central nervous system (CNS) and other neural pathways. SARS-CoV-2 infection of the GI tract and altered microbiota (dysbiosis) results in mucosal damage, infiltration of immune cells, and increased secretion and expression of cytokines (such as IL-6, IL-1β, IL-2, IL-7, IL-8, IL-9, IL-10, IL-17, tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein 1 (MCP1), macrophage inflammatory protein 1A (MIP1A), macrophage inflammatory protein 1B (MIP1B), macrophage inflammatory protein 3A (MIP3A)), IGF-1, neuroactive metabolites, 5-hydroxytryptamine, and cholecystokinin and activation of enteric nerve. These molecular events further activate the vagus nerve and disrupt the blood-brain barrier [48, 49]. Increased secretion of these cytokines may precipitate ischemia and activation of coagulation cascade causing stroke, delirium, confusion, depression, and psychological symptoms. GI infection is also associated with altered tight junction, increased mucosal permeability, entry of SARS-CoV-2 to vessels through the hepatic artery, increased vascular permeability, and leakage, thus affecting the nervous system with deficiency of serotonin and norepinephrine and higher levels of dopamine in the brain [49, 50].

There exists a bi-directional communication system between the ENS and the CNS, which suggests a pathway for the transmission of inflammatory markers and proteins from the gut to the brain. Specifically, a multi-center assessment using immunohistochemical methods revealed the presence of α-synuclein proteins in sections of the submucosa of the sigmoid colon at autopsy of Parkinson’s disease patients [51]. Additionally, the importance of the ENS can be seen with studies linking the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) with the small intestine [52, 53]. Administration of MPTP in a rat model causes loss of dopaminergic neurons in the myenteric plexus while 6-OHDA administration into the substantia nigra of the brain slows colonic motility [52, 53]. The ENS’s bi-directional communication system with the CNS and markers such as MPTP and 6-OHDA reveal the importance of studying this pathway in the pathogenesis of COVID-19-induced neurological manifestations.

SARS-CoV-2 infects human cells through its spike protein that binds to human ACE-2 receptors [54]. The ACE-2 receptor is highly expressed on the pulmonary epithelial cells, resulting in the respiratory symptoms of the COVID-19 [55]. Essential to the pathogenicity of SARS-CoV-2 is transmembrane protease serine 2 (TMPRSS2), which causes viral activation and membrane priming [56]. These proteins, (ACE-2 and TMPRSS2) play a role in GI and neurological symptoms of COVID-19 as they are also found on the epithelium of the intestines and lining of vessels of the blood-brain barrier (Figure 2A) [54, 57].

Figure 2.

The schematic diagram showing the underlying mechanism of the development of neurologic symptoms via gut in SARS-CoV-2. The SARS-CoV-2 enters the body through the nasopharynx down the esophagus into the GI system. Figure 2A: SARS-CoV-2 activation and membrane priming in the gut and brain are dependent on ACE-2 and TMPRSS2 membrane proteins on the intestinal epithelium and blood-brain barrier. Figure 2B: ACE2 activates B(0) AT1, a tryptophan transporter. Tryptophan is metabolized to form metabolites such as kynurenine (Kyn) and nicotinamide, both dependent on the rate-limiting enzyme indole 2,3-dioxygenase (IDO1). Nicotinamide activates the mTOR pathway to transcribe antimicrobial peptide agents. Kyn activates the aryl hydrocarbon receptor (AhR) found on immune cells, including CD4+ T-cells, innate immune cells, and natural killer cells. The activation of AhR leads to these immune cells secreting IL-22 cytokines to bind and activate IL-22 receptors to phosphorylate STAT3 on paneth cells in the intestinal epithelium to secrete beta-defensins and cathelicidins. Kyn also affects the microglial cells and astrocytes in the CNS to maintain the BBB and clear waste products. SARS-CoV-2 affects the whole tryptophan pathway leading to antimicrobial changes in the gut and neuroinflammation. Figure 2C: SARS-CoV-2 binds to TLR4 on intestinal epithelium to activate MYD88 to produce proinflammatory cytokines and zonulin. Zonulin is released back into the intestinal lumen to bind to protease-activated receptors-2 (PAR2). When activated, PAR2 disrupts the tight junctions in between the intestinal epithelial cells, allowing SARS-CoV-2 to enter the bloodstream. SARS-CoV-2 reaches the blood vessels in the brain to act on the TLR4 on the BBB to create more zonulin. This zonulin binds to a special zonulin receptor in the BBB that disrupts the TJ, allowing SARS-CoV-2 to enter the CNS to cause neuroinflammation.

Hyposmia and dysgeusia are the most prevalent neurological manifestations in COVID-19, with the incidence of 41.1% and 46.4%, respectively [58]. The pathogenesis of this hyposmia is explored in a study of mice which found that following intranasal inoculation of SARS-CoV-2, immunohistochemical staining showed a high density of SARS-CoV-2 antigen at the olfactory nerve after four days. In the following days, viral antigens were present in the piriform and infralimbic cortices, ventral pallidum, lateral preoptic regions, and dorsal raphe regions with first- or second-order connections to the olfactory bulb [59].

Although the olfactory center is well-established in SARS-CoV-2 neuroinvasion, it does not describe the temporal correlation of the GI and neurological symptoms. Some studies suggest that the ENS plays an important role in SARS-CoV-2 neuroinvasion [60]. A similar mechanism can be seen in HIV. For example, the colonic application of HIV-1 Tat protein, which is responsible for mucosal damage in the gut and subsequent diarrhea, causes activation of glial cells of the ENS and leads to neuroinflammation and cognitive impairment [61]. Likewise, inhibition of the glial cells of the ENS with application of HIV-1 Tat protein showed no neuroinflammation or impairments in cognitive performance, displaying the role of the glial cells ENS in viral-mediated neurologic impairment [61]. We hypothesize that SARS-CoV-2 may enter the intestinal epithelium through ACE-2 receptors and its associated proteins activate the ENS glial cells, leading to a cytokine storm and neuroinflammation.

Further supporting SARS-CoV-2 neuroinvasion via the ENS and its components, studies have demonstrated the importance of the vagus nerve and microtubules in viral infection. One experimental study unilaterally cut the vagus nerve in mice and inoculated a virulent influenza virus. Results revealed that there was viral antigen that was not strongly detected in the vagal ganglion of the vagotomized side when compared to the intact side [62]. This finding implicates the importance of the vagus nerve in viral infections.

Additionally, more research reveals that coronavirus-infected mice treated with microtubule affecting agents (vinblastine and paclitaxel) led to fewer viral antigens in the neurons [63]. Based on the findings it was hypothesized that SARS-CoV-2 disseminates into the nervous system in a retrograde fashion, with one potential entryway being the ENS and vagus nerve. These findings suggest a possible temporal association between gut-brain axis and disruption of the gut barrier but how it affects CNS remains a question because of the presence of the blood brain barrier (BBB). In the following section we discussed the aspects related to gut-associated CNS infection in the light of BBB.

4. Blood brain permeability, neuroinvasion, and COVID-19

The presence of ACE-2 in the GI tract has significant impact on the intestinal microbiota. A study in ACE-2 knockout mice revealed a decrease in the expression of antimicrobial agents which caused an alteration in the gut microbiome composition [64]. The role of ACE-2 in the small intestine is non-catalytic. ACE-2 in the lower GI stimulates the sodium-dependent neutral amino acid transporter B(0)AT1 (Figure 2B). This transporter is responsible for the transport of neutral amino acids for absorption [64]. B(0)AT1 plays a fundamental role in the absorption of amino acids including tryptophan, alanine, asparagine, and histidine [65]. Tryptophan plays a significant role in the kynurenine pathway, which is the process that converts tryptophan to nicotinamide [65]. Nicotinamide is involved in the mTOR pathway in the intestinal epithelium responsible for transcribing antimicrobial peptides [66]. With a decreased expression of antimicrobial peptides, the gut microbiota is altered.

COVID-19 has major effects on amino acid metabolism that originate in the gut. Patients who tested positive for SARS-CoV-2 showed lower levels of tryptophan than the control likely due to the decreased activity of B(0)AT1 [37, 65]. The rate-limiting step in this tryptophan pathway is indole 2,3-dioxygenase (IDO1), an enzyme with complex effects, including immunoregulation. Studies have shown an inverse correlation of IDO1 activity and severity of SARS-CoV-2, the more severe the COVID-19 infection, the lower the IDO1 levels [37]. Lower IDO1 causes many downstream effects, including a decrease in its product kynurenine that controls inflammatory responses by acting on aryl hydrocarbon receptor (AhR) [67]. Lower IDO1 and subsequent low AhR activity may explain neurological sequelae of COVID-19. AhR plays an important role in transcribing cytokines which maintain mucosal barriers and allow for the clearance of pathogens in these mucosal layers [67]. AhR is found on immune cells such as CD4+ T-cells, innate lymphoid cells (ILC), and natural killer cells (NK) [68]. When activated, AhR promotes the production of IL-22, which stimulated IL-22 receptors on paneth cells of the intestinal epithelial cells. Activated IL-22 receptors trigger STAT3 activation within the paneth cells to produce beta-defensins and cadherins [68]. These are antimicrobial agents that maintain mucosal barriers by maintaining the gut microbial composition. Lower levels of AhR and subsequently NF-κB are detrimental to astrocytes since studies have shown a direct correlation between the activity of NF-κB and astrocytes. Thus, lower levels of AhR as a direct consequence of SARS-CoV-2 action in the gut may lead to diminished activity of astrocytes via an NF-κB-dependent mechanism. Astrocytes make up the blood-brain barrier and with its suppression, protective barriers against CNS infection are lost [69, 70].

With this disruption of decreased tryptophan absorption in the gut due to SAR-CoV-2, many other endogenous pathways will be affected since tryptophan is a rate-limiting molecule in many pathways. One of the major effect is the production of serotonin and melatonin, which uses tryptophan for its synthesis [71]. There is evidence that melatonin is involved in the pathophysiology of SARS-CoV-2 infection. Indeed, melatonin has been associated with potent antioxidant and anti-inflammatory effects by activating both the cellular and humoral immune response [72, 73]. The positive effects of melatonin have been studied in other diseases with promising results to overcome cytokine storms in other virus-related illnesses [72]. The association of SARS-CoV-2 and melatonin has been noticed through a meta-analysis study that found faster recovery time in SARS-CoV-2 patients receiving melatonin early in their course of infection compared to the control group [73]. Given this information, the foundation of altered tryptophan absorption in SARS-CoV-2 infection is involved in many downstream processes.

Another mechanism by which SARS-CoV-2 in the gut alters the blood-brain barrier is through increased expression of zonulin. It is well known that the spike protein binds to the ACE-2 receptors in the intestines, however, SARS-CoV-2 can also bind to other receptors. It has been shown that the spike protein can also bind to the TLR-4 receptor on the intestinal mucosa [13]. The binding to the TLR-4 receptor causes activation of MyD-88, which is responsible for transcribing proinflammatory factor such as zonulin. Once produced, zonulin is then released into the systemic circulation and intestinal lumen [74]. In the lumen, zonulin binds to protease-activated receptors-2 (PAR2) on the apical layer of the intestinal epithelium. This binding of PAR2 disrupts the tight junctions between adjacent intestinal cells [74]. The loosening structure of the tight junction causes an increase in epithelial permeability, which will allow the SARS-CoV-2 to be transported paracellularly and enter the systemic circulation [75]. The increased zonulin protein into circulation can also bind to the zonulin receptors in the BBB. Like PAR2, the zonulin receptor disrupts the tight junctions within the BBB and results in endothelial permeability [47]. This allows for the entry of SARS-CoV-2 into the CNS. It has been shown that zonulin levels are increased in patients infected with SARS-CoV-2 [76]. The increased expression of zonulin allows the virus to enter circulation from the gut and opens the gates for SARS-CoV-2 to enter the CNS causing neuroinvasion. Thus, it is reasonable to speculate that the components of tryptophan metabolism, as well as increased zonulin play a critical role in the gut-brain axis in those with neurological manifestations of COVID-19.

The primary mode of transmission of SARS-CoV-2 is from the droplet contagion causing respiratory symptoms [77]. The mechanism of how SARS-CoV-2 invades the nervous system to cause neurologic sequelae remains unclear. Paths of neuroinvasion are still being investigated and include retrograde neural invasion and systemic invasion from blood, CSF, and lymph [40]. Here, we aim to explore routes of SARS-CoV-2 neuroinvasion via the GI tract and its components. Neuroinflammation in COVID-19 has been linked to macrophage expression myeloid-related protein 14 (MRP14) in the olfactory epithelium [78]. MRP14 initiates an inflammatory cascade via TLR-4-MyD88 signaling (Figure 2C). These MRP14 and TLR4 inflammatory processes are also evident in the GI tract. Calprotectin, also known as S100A8/S100A9, MRP8/14 (Myeloid-Related Protein), or leukocyte protein L1, is a heterodimer involved in neutrophil inflammatory processes. In COVID-19 calprotectin is associated with poor outcomes [79] Moreover, in patients with elevated calprotectin, GI symptoms were more frequent. MRP14 plays a key role in both neuroinflammation and GI severity in COVID-19.

Insulin-like-growth factor (IGF-1) belongs to an IGF family that plays a diverse role in cell proliferation, metabolism, differentiation, and endocrine control of the immune system. Evidence also supports the IGF-1 pathway to regulate immune response through interaction with cytokines, immune cells, and bone marrow cells [80]. IGF-1 regulates cell growth and apoptosis through PI3K/AKT and MAPK signaling pathways. IGF-1s play a key role in the context of influenza virus-mediated inflammation. In a study by Li et al. [81], IGF-1 affected the expression of key proteins associated with p38 in the MAPK and PI3K/AKT signaling pathways in influenza-virus mediated inflammation. IGF-1 expression and phosphorylation are upregulated following influenza virus infection, triggering PI3K/AKT and MAPK to induce inflammation.

IGF-1 also plays a critical role in viral inflammation and COVID-19-induced acute respiratory distress syndrome (ARDS). In a study by Fan et al. [82], higher IGF-1 concentrations were associated with a lower risk of COVID-19 mortality. Neurons in the hippocampal dentate gyrus and subventricular areas are regenerated through IGF-1 induction [83]. Moreover, IGF-1 plays a critical role in inhibiting immune inflammation in the mouse colon [84]. IGF-1 may serve as a key factor in protection from COVID-19-related inflammatory injury. We hypothesize that the IGF-1 pathway may reduce inflammatory mediators from crossing the intestinal barrier into the blood-brain barrier and thus decreasing the occurrence of neurological sequela.

The Th17/IL-17 axis has been linked to increased disease severity in respiratory distress and viral infections (influenza, respiratory syncytial virus) [85]. Huang et al. [86] observed that IL-17 levels are increased in intensive-care COVID-19 patients compared to non-intensive care and controls. A study by Lee et al. [87] also reveals the increased expression of IL-17 in COVID-19 patients. A Chinese clinical trial is already investigating an anti-IL-17 drug (ixekizumab) against COVID-19 [88]. Interestingly, Th17/IL-17 inflammation can be traced back to the gut whereby intestinal dysbiosis alters IL-17 and enhances neuroinflammation [45]. The pathogenesis of how the microbiota affects IL-17 producing T-cells is under investigation. In one study, it was shown that the development of Th17 cells in the intestine was influenced by IL-1β producing-microbiota [33]. The gut-brain axis can be seen at play when looking at Th17/IL17 and it is important to continue investigating how IL-17 in the gut can predict neurologic outcomes in COVID-19. Future studies are warranted to closely monitor levels of MRP14, IGF-1, and IL-17 in COVID-19 patients.

5. Biomarkers

The key biomarkers for severe SARS-CoV2 infection are shown in Table 1. These biomarkers in the blood or nasopharyngeal swab from the healthy subjects or patients with severe COVID-19 infection are derived from the studies from various investigators. It is critical to compare the findings of these biomarkers with the severity of COVID-19 infection, and the time course of the biomarker levels after initial infection. Furthermore, what is the effect of various therapies on the level of these biomarkers? Nonetheless, these biomarkers could serve a critical role in the monitoring of COVID-19 infection and the development of neurological symptoms.

Table 1.

Biomarkers for severe SARS-CoV-2 infection

Study	Biomarkers	Mechanism of action of the makers	Levels in patients with COVID- 19	Levels in the control group	p-value	Method of Collection
Giron et al, 2021	Zonulin	Zonulin increases the permeability of the intestinal epithelium barrier and blood-brain barrier by modifying tight junctions and the underlying actin cytoskeleton.	50 ng/ml	0 ng/ml	4.96e-7	Serum blood
Rossi et al, 2021	TMPRSS2/ACE2	TMPRSS2 is found on the intestinal epithelium that plays an important role in the activation and cell entry of SARS-CoV-2	0.63	0.33	<0.05	RT-qPCR from nasopharyngeal swab
Thomas et al, 2020	Tryptophan	Tryptophan is involved in the kynurenine pathway to synthesize nicotinamide, which is involved in the mTOR pathway in the intestinal epithelium. This is responsible for transcribing antimicrobial peptides	2.0 x 10-7	4.0 x 10-7	< 0.001	Serum blood
Lee et al, 2020	IL-17	IL-17 leads to endothelial dysfunction, intestinal dysbiosis, release of cytotoxic immune cell, and chemokine production in the brain tissue leading to neuroinflammation	1.225 pg/mL	0.126 pg/mL	< 0.00001	Serum blood

6. Treatment

Given the report of immune mediators and biomarkers in the gut brain-axis, in the following sections we discussed potential therapeutics based on such factors.

6.1. Probiotics

The gut microbiota is a heavily interconnected network that frames a major component of the gut-brain axis, from immune regulators, cytokines, neurotransmitters, and neurohormones.

A blinded, randomized control trial conducted on SARS-CoV-2 patients compared a four-strain probiotic with a maltodextrin carrier compared to controls with maltodextrin carrier only. The study revealed statistically significant improvement on remission rate in probiotic-treated patients compared to controls (p < 0.001). Secondary clinical outcomes reported significantly less days of fever, cough, headaches, body aches, shortness of breath, nausea, and abdominal pain. In this study, investigators concluded that the immune-stimulatory effects of probiotics influence the gut-lung axis may explain the reduced symptoms of fever, cough, and shortness of breath in this population [91]. The statistically significant reduction in headaches may be explained by the gut-brain axis but should be further studied.

Literature has shown that SARS-CoV-2 causes an increase in Ruminococcus gnavus, Ruminococcus torques, and Bacteroides sp. These bacteria associate with other GI pathologies such as inflammatory bowel disease and ulcerative colitis causing gut dysmotility [92]. There are other bacterial species that have been shown to be beneficial to the gut to help defend against SARS-CoV-2. There have been reports of improved clinical outcomes of SARS-CoV-2 patients with F. prausnitzii, B. dorei, Bacteroides thetaiotaomicron, Bacteroides massiliensis, and Bacteroides ovatus in their gut [93]. Researchers have shown Bacteroides causes positive outcomes for SARS-CoV-2 by down-regulating the ACE-2 receptors in the gut [92]. This ultimately prevents the GI invasion of the virus, thus preventing it from invading the nervous system.

Gut microbiota influences these routes of communication and influences the CNS through the secretion of microbial products and metabolites. For instance, the microbiota enhances the blood-brain barrier by increasing tight junction proteins and decreasing permeability [87]. Furthermore, studies in antibiotic-treated mice reveal an adverse effect on neurodevelopment and neurodegenerative disorders [94]. Extensive literature has been reported on the efficacy of probiotics in various neurologic disorders such as autism spectrum disorder and neurodegenerative diseases [36, 95]. With the enhancement of gut immunity, probiotics can also mitigate the neurologic effects down the line.

6.2. Interleukin-17

In this section, we bring back the importance of IL-17 in the gut-brain axis, COVID-19 cytokine storm, and potential treatment modalities to target IL-17 in the treatment of neurologic symptoms in COVID-19. IL-17 is a proinflammatory cytokine that is implicated in the inflammation seen in COVID-19 [85]. One meta-analysis on IL-17 levels in COVID-19 patients revealed an increase in IL-17A levels in COVID-19 patients in relation to disease severity [96]. A study on the course of COVID-19 in patients with psoriasis treated with secukinumab was very mild [97, 98]. Another study revealed that secukinumab lowered ACE-2 expression on the skin of psoriatic patients [97, 98]. Such findings in the treatment of COVID-19 with secukinumab coupled with the importance of IL-17 in the gut-brain axis warrant future investigations in targeting IL-17 in COVID-19 patients with neurologic symptoms.

6.3. TMPRSS2 inhibitors

As mentioned above, the membrane protein TMPRSS2 found on the intestinal epithelium plays an important role in the activation and cell entry of SARS-CoV-2 [56]. Inhibition of the TMPRSS2 can prevent the virus entry into intestinal epithelium, upper respiratory tract, and BBB preventing the disease severity to develop [99]. Several TMPRSS2 inhibitors have been discovered for use against SARS-CoV-2 such as camostat mesylate and nafamostat mesylate (Figure 3A) [100]. An ex-vivo experiment using recombinant forms of TMPRSS2 from human lung tissues was cultured and plated with Camostat mesylate and nafamostat mesylate and was found to block the activity of TMPRSS2 [101]. Another study used mice that underwent transduction with human ACE2 mimicking human airway epithelia that were infected with SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV). They found that Camostat mesylate and nafamostat mesylate both reduced weight loss, viral burden, and mortality in the transgenic mice for both coronavirus infections [102]. This study signifies that the TMPRSS2 inhibitors can be used as a potential treatment modality. Currently, clinical trials are investigating the efficacy of Camostat mesylate and nafamostat mesylate for their use against COVID-19 patients [103, 104]. The use of these inhibitors can prevent the viral activation and cellular entry of the virus, therefore preventing severe infection.

Figure 3.

The mechanisms of different treatment options to help prevent severe SARS-CoV-2 infection. Figure 3A: TMPR22 inhibitors such as camostat mesylate and nafamostat mesylate block the action of TMPRSS2, preventing viral activation and membrane binding. This prevents SARS-CoV-2 replication in the intestinal epithelium and prevents SARS-CoV-2 from reaching the bloodstream to reach the CNS. Figure 3B: Nicotinamide supplements replenish the levels caused by the decreased tryptophan absorption from SARS-CoV-2 leading to transcription of antimicrobial agents. With decreased Kyn from SARS-CoV-2, AhR agonists such as pelargonidin, FICZ, and OMP can be used to activate AhR receptors on the immune cells to secrete IL-22. This allows increased secretion of beta-defensins and cathelicidins from paneth cells on the intestinal epithelium. Nicotinamide and AhR agonists both maintain the gut microbial composition preventing a cytokine storm of proinflammatory markers. Figure 3C: SARS-CoV-2 increases the zonulin levels leading to tight junction (TJ) disruption. Larazotide acetate (AT1001) is used to prevent the binding of zonulin from PAR2 in the intestinal epithelium, blocking the disruption of the TJ that zonulin causes. This prevents SARS-CoV-2 from entering the bloodstream.

6.4. Nicotinamide

With the SARS-CoV-2 binding of ACE-2 inhibiting the action of B(0)AT1, patients with the infection have decreased tryptophan absorption. This leads to many downstream effects such as decreased nicotinamide levels or decreased AHR activation (Figure 3B). Replenishing patients with the downstream metabolite of nicotinamide may help with COVID-19 infections. When the vitamin B3 derivative, nicotinamide, is co-incubated whole human blood mixed endotoxins, pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNFα were reduced [105]. This in-vitro study indicates human inflammatory disease can be reduced with nicotinamide supplementation. When studied in human trials that administered a supplement of combined metabolic activators that consisted of nicotinamide riboside, there was a significant reduction in recovery time compared to placebo in patients infected with SARS-CoV-2 (5.7 vs 9.2 days) [106]. Plasma levels from these patients showed that patients taking the combined metabolic activators had significantly improved inflammation with decreased levels of cytokine levels such as IL-6, IL-10, IFN-γ, and TNFα [106]. There is currently a clinical trial investigating 1 gram of nicotinamide riboside given orally for 14 days to reduce the severity of COVID-19 infection in elderly patients [107]. Given the clinical data that nicotinamide can reduce inflammation, it could be considered as a treatment modality for COVID-19.

6.5. AhR agonists

Another metabolite of tryptophan metabolism is kynurenine, which is used to activate AhR. Activation of AhR leads to the release of anti-inflammatory cytokines that helps prevent inflammation. From the reduction of tryptophan absorption in SARS-CoV-2, there is reduced kynurenine to allow AhR activation. The use of an AhR agonist may help with COVID-19 infections to increase anti-microbial agents to maintain the gut microbiome. The use of the AhR agonist, pelargonidin, in mouse models showed decreased intestinal inflammation with reduced levels of IL-1, IL-6, IL-8, and TNF-α [92]. Pelargonidin decreases the expression of ACE-2 thereby directly inhibiting the binding of SARS-CoV-2 to cells (Figure 3B) [108]. Another study used other AhR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) against mammalian cells infected against SARS-CoV-2. They demonstrated that FICZ and OMP lead to decreased expression of ACE2 leading to suppression of SARS-CoV-2 infection [109]. This evidence is a significant to proof of a positive outcome for the use of AhR agonist against COVID-19 infections. Human trials against these medications are warranted before their consideration as a treatment modality.

6.6. Zonulin

Zonulin increases in patients with severe infection of SARS-CoV-2 leading to increased intestinal membrane and BBB permeability. Inhibition of zonulin to prevent increased permeability is another mechanism to help prevent severe infections of SARS-CoV-2. Larazotide acetate (AT1001) is a synthetic amino acid peptide zonulin inhibitor that functions as a tight junction regulator, which is currently being clinically studied for treatment in patients with celiac disease (Figure 3C) [110]. Preventing the action of zonulin with AT1001 has been shown in many inflammatory diseases such as arthritis and Crohn's disease to restore the impaired intestinal barrier [89]. Additionally, it was discovered that AT1001 also binds to the catalytic domain of the SARS-CoV-2 main protease (Mpro), which is an enzyme that is essential to viral replication [110, 111]. The use of AT1001 has been clinically studied for multisystem inflammatory syndrome in children (MIS-C) that was caused by SARS-CoV-2. The children with MIS-C from SARS-CoV-2 showed high levels of zonulin leading to the increased presence of SARS-CoV-2 in the bloodstream [112]. These patients who were treated with AT1001 showed a substantial decrease in SARS-CoV-2 Spike protein antigen levels and other inflammatory markers such as IL-17, IL-2, IFN-γ, and IL-1 [112]. By blocking the viral replication through Mpro and preventing the action of zonulin, it leads to fewer SARS-CoV-2 particles replicating and entering the bloodstream through the intestinal barrier. Although there is an active clinical trial for AT1001 for MIS-C, more clinical studies are needed to investigate the effects of AT1001 in adults infected with COVID-19.

7. Conclusion

Extensive reports of the etiologies of SARS-CoV-2 infection present a complex understanding of the factors involved, causes of the disease, and symptoms. It is understood that the major clinical manifestations of COVID-19 involve the respiratory system, but the key factor in the pathogenesis is related to the immune system affecting other organs through paracrine and endocrine effects of the secreted cytokines and hormones [114, 115]. Retrograde transmission of the virus from the ENS, microbiota, and cytokines into the CNS are evident in the literature. Such immune factors and biomarkers of the gut-brain axis contribute to increased expression of cytokines, increased permeability of the blood-brain barrier, and ultimately injury to the CNS, which contribute to COVID-19 related neurological symptoms. This review critically summarized important biomarkers in the gut-brain axis. The advances made in our understanding of the pathogenesis of COVID-19 neurologic sequelae could serve as a guide for diagnosis, prevention, and treatment of COVID-19-related neurologic effects. The key practical message of this article is that it is critical to screen SARS-CoV-2 patients with GI symptoms for neurological manifestations.

This article also summarized the possible underlying molecular mechanisms for gut-brain communication and neurological sequalae, however, there is a need for further discussion and list the differential neurological findings in patients with GI symptoms in various studies. Further, the sensitivity, specificity, and accuracy of the GI biomarkers associated with CNS manifestations must be examined and reported. The biomarkers listed here are not an exhaustive list and warrant future studies to analyze other biomarkers as well as their accuracy in the setting of the gut-brain axis and SARS-CoV-2 infection.

8. Expert opinion

Gut infection with SARS-CoV-2 and inflammation cause altered gut microbiota and increased anti-microbial agents in GIT. This causes altered expression of various mediators and increased secretion of zonulin which cause disrupted tight junction. All these events stimulate enteric nervous system and signals to central nervous system precipitating neurological sequalae. Most of the research relating to the treatment of SARS-CoV-2 focuses on treating symptoms and the respiratory route of entry. However, fecal-oral route of entry and GI pathogenesis has not been thoroughly investigated. The available literature suggests potential role of B^OAT1 (SLC6A19), ACE2, TMRSS2, TMPRSS4, IFN-γ, IL-17A, zonulin, and altered gut microbiome in gut-brain axis and associated neurological sequalae. Thus, targeting these mediators and gut microbiome will be of therapeutic significance. The role of probiotics in the treatment of COVID-19 has been discussed in the literature but the role of targeting IL-17, TMRSS2, TMPRSS4, IFN-γ, and supplementing nicotinamide for the treatment of COVID-19 has not been investigated in-depth. Most of the available literature discussing the potential of targeting IL-17, TMRSS2, TMPRSS4, IFN-γ, and supplementing nicotinamide are review articles and thus well-designed clinical trials are warranted.

Further, cytokine storm plays a critical role in COVID-19 pathogenesis and targeting TNF-α might be a potential strategy to treat COVID-19. The paucity of available literature on targeting TNF-α suggest the significance of investigating TNF-α as a therapeutic target. It will be of interest to investigate whether targeting TNF-α receptors or soluble TNF-α will be of higher therapeutic potential. Similarly, investigating IFN-γ as a potential target should be considered. Targeting IL-6 in COVID-19 is under research but targeting oncostatin M (OSM) has not been investigated. Targeting OSM, a pleiotropic, interleukin-6 family inflammatory cytokine, will be of importance because increased levels of OSM has been reported in COVID-19 patients. Further, immune cells secreting inflammatory cytokines play a critical role in COVID-19 pathogenesis and targeting immune cell response may be another therapeutic strategy. Few reports suggest that CAR-T cell therapy associate with poor outcome, but CAR-NK cell therapy is beneficial in effectively treating SARS-CoV-2 spike protein in-vitro and thus should be deeply investigated. Attenuated immunity is a risk factor for COVID-19 infection, boosting immunity and strategies to improve immunity will be of significance. Additionally, a panel of biomarkers including cytokine levels in at risk patients might be of importance to evaluate the gut immunity in at risk patients because poor gut immunity will increase the risk of fecal-oral transmission.

In-depth understanding of the gut-brain temporal relationship and the underlying pathophysiology become critical with the evolving evidence in the increasing published literature reporting long-term sequalae and manifestations of SARS-CoV-2. Additionally, the presence and association of GI symptoms with neurological symptoms, positive findings of oral-GI, olfactory-GI, and lung-GI axis and associated symptoms in COVID-19 patients emphasize the need of investigating temporal relationship between different organ systems. Evolving phenotypes of COVID-19 virus and evolving concept of Long COVID syndrome further compel us to investigate the underlying pathophysiology and develop better therapeutics. The need of further research is also supported by the fact that even vaccinated people are getting infected with COVID-19, though will less severe symptoms.

In summary, a collaborative and multispecialty approach is a must to treat COVID-19 because SARS-CoV-2 target and affect multiple organs including GI tract. This is of utmost importance because changing variants of SARS-CoV-2 are present and investigating these aspects are expected to have better therapies available for COVID-19 in next five year.

Article highlights.

• SARS-CoV-2 has acute and long-term neuropsychological sequalae.

• SARS-CoV-2 enters human body through respiratory and fecal-oral route.

• ACE-2 plays a critical role in the entry and pathogenesis.

• Gut-brain axis plays a crucial role in ensuing neurological symptoms.

• B^OAT1 (SLC6A19), ACE2, TMRSS2 and TMPRSS4 play a role in viral entry and IFN-γ, IL-17A and zonulin play a role in pathogenesis of symptoms associated with gut-brain axis.

• Targeting these mediators might be of therapeutic importance to reduce long-term neurological symptoms.

Abbreviations:

ACE-2

Angiotensin-converting enzyme-2

Ahr

Aryl hydrocarbon Receptor

AT1001

Larazotide acetate

BBB

Blood Brain Barrier

CNS

Central nervous system

Th

CD4+ T-Helper cells

COVID-19

Coronavirus-19

ENS

Enteric nervous system

FICZ

6-Formylindolo(3,2-b)carbazole

GALT

Gut-associated lymphatic tissue

HIV

Human immunodeficiency virus

6-OHDA

6-hydroxydopamine

5-HT

5-hydroxytryptamine/Serotonin

IDO1

Indole 2,3-dioxygenase

ILC

Innate lymphoid cells

IGF-1

Insulin-like growth factor

IL-17

Interleukin-17

IFN

Interferon

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MERS-CoV

Middle East respiratory syndrome coronavirus

MAPK

Mitogen-activated protein kinase

MIS-C

Multisystem inflammatory syndrome in children

MYD88

Myeloid differentiation primary response 88

MRP14

Myeloid-related protein 14

NK

Natural killer cells

NF-κB

Nuclear Factor-κB

PD

Parkinson’s disease

PNS

Peripheral nervous system

PI3K

Phosphoinositide 3-kinases

PAR-2

Protease-activated receptors-2

Mpro

SARS-CoV-2 main protease

SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2

B(0)AT1

Sodium-dependent neutral amino acid transporter (SLC6A19)

TLR

Toll-like receptor

TMPRSS2

Transmembrane protease serine 2

Data availability

Not applicable; all information is gathered from published articles.