Gut microbiome and anti-viral immunity in COVID-19

Abstract SARS-CoV-2 mainly affects the respiratory system, but the gastrointestinal tract is also a target. Prolonged gut disorders, in COVID-19 patients, were correlated with decreased richness and diversity of the gut microbiota, immune deregulation and delayed viral clearance. Although there are no definitive conclusions, ample evidence would suggest that the gut microbiome composition and function play a role in COVID-19 progression. Microbiome modulation strategies for population stratification and management of COVID-19 infection are under investigation, representing an area of interest in the ongoing pandemic. In this review, we present the existing data related to the interaction between gut microbes and the host’s immune response to SARS-CoV-2 and discuss the implications for current disease management and readiness to face future pandemics.


Introduction
Coronavirus disease 2019 , caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was declared a pandemic by the World Health Organization in March 2020 (Yamamoto et al. 2021).SARS-CoV-2 infection is characterized by a broad range of clinical manifestations, from asymptomatic to severe pneumonia and mortality, especially in elderly and/or immunocompromised subjects (Abid, Nunley, and Abid 2020;Jordan, Adab, and Cheng 2020;Ragab, et al. 2020;Zhou, Yu, et al. 2020;Hoong et al. 2021).Underpinning the mechanisms responsible for the different host's immune response triggered by SARS-CoV-2 is of key importance in the fight against COVID-19 and would increase our readiness to face future pandemics (Figure 1).The lungs are the primary organ affected by COVID-19 disease.However, dysfunction of the intestine, liver or multiple organ failure have also been reported (Chen et al. 2020;D' Amico, et al. 2020;Pan et al. 2020).Evidence suggests that the gastrointestinal tract (GIT) might play a role in the COVID-19 pathogenesis.For example, SARS-CoV-2 can replicate in small intestinal enterocytes and viral RNA can be detected in fecal samples of SARS-CoV-2 infected patients that present an altered gut microbiota (Gu, Han, et al. 2020;Lamers et al. 2020;Wolfel, et al. 2020;Zuo, Zhan, et al. 2020;Cheung et al. 2022).In the intestine the virus alters the natural innate immune response and impairs the IFN autocrine action in SARS-CoV-2-infected enterocytes, promoting its replication and representing a pro-inflammatory reservoir as shown in organoid studies (Triana et al. 2021).The GIT is home to approximately 10 14 bacteria that modulate the host's immune responses (Schirmer et al. 2016).Therefore, even though no specific interactions between the gut bacteria and SARS-CoV-2 have been identified so far, the intestinal microbiota can account for differences in the host's immune responses to COVID-19 (Han, Duan, et al. 2020;Troisi et al. 2021;Yeoh et al. 2021).These alterations may affect viral infectivity through direct or indirect mechanisms (Pfeiffer and Virgin 2016;Li, et al. 2019).The microbiota can promote viral infectivity, for example, (i) facilitating genetic recombination and generation of diverse and resistant progenies; (ii) contributing to the viral stability through interactions with bacterial structural components (i.e., peptidoglycan or lipopolysaccharide); (iii) facilitating the differentiation or attachment to host-cell targets; (iv) stimulating the lytic reactivation of the virus via a short chain fatty acids (SCFA)-mediated mechanism; and (v) suppressing or activating the local host immune responses to the viral infection.In addition, it is well known that the gut microbiota affects the immunity and inflammation in the lungs of several respiratory illnesses (McAleer and Kolls 2018;Dang and Marsland 2019).However, very little is known about the gut-lung crosstalk and the associations of gut microbiota features with COVID-19 susceptibility and severity.

Gut microbiome
The human microbiome is defined as the pool of microbial genes and microbial cells that naturally live in and on our body.The microbiota is the collection of microbes inhabiting a specific niche such as the gut.The gut microbiota constitutes the most complex, dynamic, and heterogeneous ecosystem of the human body, integrated by trillions of microbes that interact with one another, and play a key role in regulating the host's physiology and, specifically, immunity (Chen, et al. 2021;Hussain et al. 2021).The eubiotic microbiota contains more than 100 bacterial phyla, mainly belonging to Firmicutes and Bacteroidetes and, secondarily, to Actinobacteria, Proteobacteria, Fusobacteria and Verrucomicrobia.The gut microbiota and the human body are in a symbiotic relationship which benefits the survival and functional competencies of both organisms (Nicholson et al. 2012;Francino 2014).Specifically, the host's immune system interacts with the microbiota contributing to intestinal homeostasis by tolerating symbiotic bacteria and, at the same time, fighting invasive pathogens (Malard et al. 2021).Alteration of the gut microbiota and its functionality (e.g., reduced diversity, increased abundance of pathobionts, reduced potential for SCFA production, etc.) is known as dysbiosis and often associated with disorders underlying inflammation and immune dysregulation such as inflammatory bowel disease, diabetes, coronary heart disease, neurodegenerative diseases, depression, and infections, among others (Dinan and Cryan 2017;Al-Rubaye, Perfetti, and Kaski 2019;Gurung et al. 2020;Bastiaanssen and Cryan 2021).Dysbiosis can contribute to and be the result of perturbation of the homeostatic balance between the microbiome and the host (disruption of the symbiosis), leading to parallel alterations in host's functions, such as loss of the natural intestinal permeability and immune control and, ultimately, contribute to several disorders, from diarrhea and constipation, and to chronic inflammatory diseases (Belizario and Faintuch 2018).Evidences suggest that the commensal gut microbiota plays immuno-stimulatory or suppressive roles in viral infections contributing to disease susceptibility and severity.In turn, the gut microbiota can be disturbed by viral infections and, thereby, contribute to viral infectivity and evolution, being involved in a causality circle.

The gut bacteriome in SARS-CoV-2 infection
It is estimated that over 60% of COVID-19 patients have gastrointestinal symptoms, including diarrhea, nausea, and vomiting (Chen et al. 2020;Jin et al. 2020;Liang et al. 2020;Tian et al. 2020;Zhang et al. 2020;Groff et al. 2021).Gut microbiota alterations are shown in both patients with and without gastrointestinal symptoms and persist even after clearance of SARS-CoV-2 in the respiratory tract (Neurath, Überla, and Ng 2021).Also, SARS-CoV-2 seems to be capable of infecting intestinal bacteria in vitro, showing a bacteriophage-like behavior.This might increase the SARS-CoV-2 mutability and explain the persistence of SARS-CoV-2 in feces of recovered patients (Petrillo et al. 2021).However, the normally large inter-individual variation and the multiple interacting host and environmental factors affecting the gut microbiota have probably prevented the identification of the consensus COVID-19-microbiota signatures linked to specific COVID-19 pathogenic features (Petrillo et al. 2021;Zuo, Liu, et al. 2021).Table 1 summarized the most relevant studies done in the field.The COVID-19 gut microbiota alterations identified so far include depletions of commensal bacteria from the families Ruminococcaceae and Lachnospiraceae (Gu, Han, et al. 2020), some of which are important butyrate producers which may help strengthen the gut barrier and maintain immune homeostasis (Vacca et al. 2020).Also, bacterial species with potential immunomodulatory properties, such as Faecalibacterium prausnitzii, Eubacterium rectale, and several Bifidobacterium species are diminished in COVID-19 patients (Gu, Han, et al. 2020;Yeoh et al. 2021;Farsi, et al. 2022).Bacterial alterations persist over the course of COVID-19 and can remain significantly altered for up to 6 months after clearance of SARS-CoV-2 from the  respiratory tract (Yeoh et al. 2021;Chen et al. 2022).For example, microbiota richness was not found to be restored in COVID-19 patients after a 6-month recovery period and this feature was related to higher C-protein levels during the acute phase of the infection and worse pulmonary functions during the postconvalescence phase (Chen et al. 2022).Some COVID-19 patients, even after clearance of the SARS-CoV-2, develop fatigue, headache, attention disorder, hair loss, shortness of breath, dyspnea and joint pains, a phenome called long-COVID (Liu et al. 2022) The exact cause of long-COVID is still unknown and every person who has a COVID-19 infection runs the risk of developing long-term symptoms.However, the gut microbiota might play a key role in the development of symptoms associated with the long-COVID (Chen et al. 2022).The microbiota of patients who recovered from infection, but did not have long-COVID, had fully recovered after six months.In contrast, people with long-COVID had a significant reduction in bacterial species and different gut microbiota depending on their symptoms (Chen et al. 2022).The SARS-CoV-2 infection has also been associated with alterations in the microbiome functional capacity including increases in nucleotide de novo biosynthesis, amino acid biosynthesis and glycolysis (Zuo, Liu, et al. 2021).A correlation has been shown between the depletion of several bacterial species in COVID-19 patients and the increase in sera concentrations of pro-inflammatory cytokines and chemokines.These include tumor necrosis factor (TNF)-α, C-X-C motif chemokine ligand (CXCL)-10, proB-type natriuretic peptide, C-reactive protein, monocyte chemoattractant protein-1 (MPC-1), interleukin(IL)-6 and IL-10, indicating that these depleted taxa may play a role in preventing excessive inflammation (Zeng et al. 2016;Han, Duan, et al. 2020;Vabret et al. 2020).Additionally, some of the depleted gut commensals have previously been shown to evoke protective responses, stimulating defensive inflammatory immune responses or reducing excessive inflammation by promoting the expression of anti-inflammatory cytokines and T regulatory (T reg ) cells (Riedel et al. 2006;Cattaneo et al. 2017;Alameddine et al. 2019).The depletion of commensals is linked to the enrichment of potential pathobionts (such as Ruminococcus gnavus and Ruminococcus torques, Bacteroides nordii and Bacteroides vulgatus, Clostridium hathewayi and Actinomyces viscosus) and to a significantly decreased species diversity (Tang et al. 2020;Zuo, Zhan, et al. 2020;Yeoh et al. 2021).Although the differentiation between commensals and pathobionts requires a much deeper characterization, R. gnavus and R. torques have been linked to inflammatory bowel disease, Bacteroides dorei and Bacteroides vulgatus have been implicated in ulcerative colitis, Clostridium hathewayi has been involved in various infections linked to fatal clinical cases (including sepsis) and Actinomyces viscosus has been correlated to lung infections (Eng et al. 1981;Linscott et al. 2005;Davis-Richardson, et al. 2014;Matsuoka and Kanai 2015;Hall et al. 2017).A high abundance of SCFA-producing bacteria (Alistipes onderdonkii, Parabacteroides merdae, and Lachnospiraceae bacterium) has been linked to patients with low SARS-CoV-2 infectivity (Zuo, Liu, et al. 2021).Interestingly, Alistipes onderdonkii is considered a key player in maintaining gut homeostasis and has been negatively associated with COVID-19 severity (Gao, et al. 2018;Zuo, Zhan, et al. 2020).Increased gut permeability and microbial translocation have been associated with the hyper-inflammatory response of COVID-19 patients (Giron, et al. 2021).Another study reported reductions in butyric a c i d -pro du c i n g b a c t e r i a a n d i n c re a s e s i n lipopolysaccharide-producing bacteria in COVID-19 patients compared to controls (Ren et al. 2021).Stability of gut microbiota composition has also been associated with better disease progression in hospitalized COVID-19 patients and differences in composition were related to disease severity, being larger in severe and fatal cases (Schult et al. 2022).However, it is still unknown whether the viral infection causes the blooming of the inflammatory-associated gut microbes contributing to COVID-19 severity or, by contrast, a permissive microbiota depleted of commensals facilitates the viral infection, or both.More recently, the gut microbiota composition and function have been linked to the immune response to COVID-19 vaccines.In particular, Bifidobacterium adolescentis and pathways related to carbohydrate metabolism positively correlated to high neutralizing antibodies to CoronaVac vaccine (Ng, Peng et al.).The findings indicate that gut microbiota might act as an adjuvant in COVID-19 vaccination, which could be of significance specially for low vaccine responders, such as obese and diabetic patients and the elderly.

The gut mycobiome in SARS-CoV-2 infection
The mycobiome, the fungal community that lives in the gut involved in bacteriome assembly and immune development (van Tilburg Bernardes et al. 2020), is also altered in COVID-19 subjects.For example, there is an increase in opportunistic fungal pathogens, such as Candida albicans, Candida auris and Aspergillus flavus during the viral infection (Zuo, Zhan, et al. 2020).Candida albicans has been shown to impair gut bacteriome assembly even after disruption by antibiotics and inflammation (Zuo et al. 2018).Additionally, the growth of certain bacterial genera, such as Klebsiella and Escherichia, is inhibited by Candida albicans (Rao et al. 2021).Notably, fungal pathogenic species associated with pneumonia and respiratory symptoms, such as Aspergillus flavus and Aspergillus niger, were detected in fecal samples of patients even after clearance of SARS-CoV-2 infection (Zuo, Zhan, et al. 2020).These data suggest gut mycobiome alterations in COVID-19 are related to systemic dysregulation of host immunity (Zuo, Liu, et al. 2021).However, further studies are needed to investigate the long-term effect of gut mycobiome alterations in SARS-CoV-2 infection.

Gut microbiome and angiotensin-converting enzyme 2 in SARS-CoV-2 infection
Entry into host cells is the first step of any viral infection (Figure 1).The angiotensin-converting enzyme 2 (ACE2) is the binding site of SARS-CoV-2 for host entry (Zhou, Yu, et al. 2020).Membrane fusion of the virus and the host cell is activated after binding, and viral RNA is subsequently released into the cytoplasm, establishing infection.The transmembrane serine protease 2 (TMPRSS2) is an important priming enzyme that plays a major role during this process (Hoffmann et al. 2020).ACE2 is expressed in several human tissues/organs.In the respiratory system it is mainly expressed on type II alveolar epithelial cells, confirming that the lungs are the primary target of SARS-CoV-2 (Gheware et al. 2022).There is also some evidence that ACE2 is expressed in the stomach, ileum, and colon at higher levels compared to the lungs (Liang et al. 2020).Co-expression of ACE2 and TMPRSS2 genes has been detected by single-cell RNA-sequencing analyses on goblet secretory cells (nasal mucosa), type-2 pneumocytes (lungs), and absorptive enterocytes (small intestine), suggesting several potential entries of SARS-CoV-2 (Ziegler et al. 2020) (Figure 1).ACE2 was also demonstrated to regulate amino acid transport, expression of antimicrobial peptides, microbial ecology, and inflammation in the gut (Hashimoto et al. 2012).The aforementioned functions are suggested to be regulated upon SARS-CoV-2 attachment to ACE2 receptors on human small intestine enterocytes (Lamers et al. 2020;Shang et al. 2020).Interestingly, studies on the murine gut have shown that bacterial species from the Bacteroidetes and the Firmicutes phyla can down-regulate ACE2 expression (Geva-Zatorsky et al. 2017).Zuo T. and colleagues have observed that Bacteroidetes abundance in the gut microbiome of COVID-19 patients was inversely correlated with symptoms severity.Among these species, Bacteroides dorei was previously shown to inhibit colonic ACE2 expression in a murine model (Geva-Zatorsky et al. 2017;Zuo, Liu, et al. 2021).Patients with a preexisting chronic condition (such as diabetes and obesity among others) were characterized by low abundance of Bacteroides species and with the highest COVID-19 mortality rate (Fang, Karakiulakis, and Roth 2020).Collectively, these data imply a potential relationship between SARS-CoV-2, gut microbiome, ACE2 expression; moreover, host immunity, may underlie the varying anti-SARS-CoV-2 immune responses although co-morbidities could also act as confounders.

Gut microbiome and immune regulation of SARS-CoV-2 infection
Multiple sources of evidence suggest that the gut microbiota plays a key role against viral infections by modulating antiviral innate and adaptive leucocyte function in the host immune system (de Vrese, Rautenberg, et al. 2005;Rooks and Garrett 2016;Thackray et al. 2018;Trompette et al. 2018;Graversen, et al. 2020;Yaron et al. 2020).The modulation of type I IFN response is one of the mechanisms by which the gut microbiota influences the host's antiviral response (Wirusanti, et al. 2022).Type I IFNs (namely IFN-α and IFN-β) are a class of cytokine, secreted by host cells upon viral or bacterial infection, that have a potent antiviral activity (McNab et al. 2015).The role of IFN signaling in response to bacteria is yet to be elucidated and seems to be potentially mediated by direct and indirect host-microbe interactions.Conserved microbial molecules (i.e., bacterial flagellin) are recognized by Toll-like receptors (TLRs), a family of cell surface or endosome receptors (Kumar et al. 2009;Takeuchi and Akira 2010).TLRs 2, 3, 4, 7, 8 and 9, which recognize bacterial products, have been linked to the induction of type I IFNs (Monroe, McWhirter, and Vance 2010).TLR3 and 4 signaling induce type I IFNs in many cell types, while TLR2,7,8 and 9 do so in specialized cell types (plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) (Sariol and Perlman 2021).Commensal bacteria maintain type I IFNs expression at a very low level (basal level), which is necessary to trigger a proper antiviral immune response (Ganal et al. 2012;Schaupp et al. 2020).Germ-free (GF) mice show an altered basal level of type I IFNs leading to defective or delayed viral clearance following infection (Ganal et al. 2012;Schaupp et al. 2020).It has been shown that the gut microbiota plays a role in regulating the type I IFNs produced by pDCs, required for the transcriptional programming of cDCs (Schaupp et al. 2020).Indeed, cDCs, isolated from GF mice, lack the transcriptionally active regions for type I IFNs (Ganal et al. 2012).In microbiota depleted mice, macrophages showed a downregulation in IFNs genes and these animals were more susceptible to lymphocytic choriomeningitis virus and influenza virus, indicating that even macrophages depend on the microbiota to produce IFNs (Abt et al. 2012).The reduced level of basal IFNs leads to an impaired induction of adaptive immune response when mice were infected with the viruses.Microbiota-depleted mice infected with encephalo-myocarditis virus (EMCV) showed impaired natural killer (NK) cell toxicity and decreased type I IFNS expression.However, EMCV replication in brain cells was inhibited in conventional mice, suggesting a role of gut microbiota in influencing basal level of type I IFN responses and viral control at extraintestinal sites (Yang et al. 2021).A recent study in murine models also showed that gut microbiota primes the IFN-I system and, thereby, the anti-viral response through membrane vesicle-mediated delivery of bacterial DNA into host cells, located remotely without the involvement of TLRs interactions (Erttmann et al. 2022).Patients with severe COVID-19 have been shown to have a delay and/or a defective type I IFNs response leading to lung damage, together with ineffective anti-SARS-CoV-2 antibodies production (Figure 2) (Combes et al. 2021;Merad, Subramanian, and Wang 2021;Pairo-Castineira et al. 2021).However, minimal or no beneficial effect has been shown using IFNs therapies in patients hospitalized with COVID-19 (Hung et al. 2020;Pan et al. 2020;Feld et al. 2021;Monk et al. 2021).Additionally, the use of exogenous IFNs might be ineffective against COVID-19 infection (Wirusanti, et al. 2022).The benefits derived from these therapies may depend upon appropriate timing of administration and their use in the suitable patient populations (Park and Iwasaki 2020).The gut microbiota could affect COVID-19 infection locally in the gut by stimulating the host's innate immune response to SARS-CoV-2 through optimal activation of IFNs, blocking the viral replication and spread.In fact, SARS-CoV-2 can infect various tissues of the gastro intestinal tract followed by intestinal cell death and release of various pro inflammatory cytokines to compromise the intestinal barrier (Jiao et al. 2021).Theoretically, the IFNs-priming effect of the commensal microbiota could also extend from the gut to extra-intestinal sites.Therefore, using microbiota-dependent induction of endogenous IFNs responses could be a strategy to protect or treat patients affected by severe COVID-19 infection.Further understanding of how the gut commensals control the IFNs responses might be key to generating novel antiviral protective strategies based on this principle.
Inflammasomes have been described recently and related to COVID-19 infection.The inflammasome is a cytosolic multiprotein complex that plays a crucial role in inflammation and cell death.The sensor proteins in the inflammasome complex detect various microbial and endogenous stimuli, leading to subsequent caspase activation.The activation of caspases results in the maturation of the pro-inflammatory cytokines IL-1β and IL-18 or pyroptosis (a mechanism of cell death) (Watanabe, Guo, and Kamada 2021).The interactions between the gut microbiota and the inflammasome play crucial roles in the gastrointestinal tract.The gut microbiota induces the expression and activation of inflammasome proteins, which contribute to both homeostasis and disease (Schroder and Tschopp 2010;Vanaja, Rathinam, and Fitzgerald 2015).Inflammasomes have mainly been studied in monocytes and macrophages, however, recent evidence suggest that they play a key role in the release of neutrophil extracellular traps (NETs) by neutrophils in patients affected by severe COVID-19 (Aymonnier et al. 2022).The excessive accumulation of neutrophils and NETs in the SARS-CoV-2 infected lung may contribute to increased mucous viscosity, which in turn may impair ventilation (Linssen et al. 2021).Targeting inflammasome to reduce NETs could restore homeostasis.Additionally, it has been suggested a role of SARS-CoV-2 infection in inducing pyroptosis in peripheral blood mononuclear cells through NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, cleavage of caspase-1, and secretion of IL-1β and IL-18 (Rodrigues, et al. 2021).Specially, interactions between the NLRP3 activation and the gut microbiota have been described and related to various diseases, such as inflammatory bowel diseases and atherosclerosis, but evidence connected to COVID-19 is very limited.In this regard, a small-scale study reported that COVID-19 patients with cardiac problems had elevated markers of a leaky gut, including the lipopolysaccharide-binding protein, and of inflammasome activation, suggesting a role of gut microbial products leaked in subsequent inflammasome activation in COVID-19 with comorbidities (Hoel et al. 2021).A potential role of probiotic bacteria in treating COVID-19 infection via NLRP3 regulation (inhibition or activation) has been discussed but not proved in clinical trials so far.
The gut microbiota also plays a role in regulating adaptive immunity and, thereby, the activation of anti-viral defence mechanisms, including the induction of antibody-producing B-cells and the effector CD4 (helper) and CD8 (cytotoxic) T cells that generate pathogen-specific memory.GF and antibiotic-treated mice have reduced levels of intestinal immunoglobulin (Ig) A producing B cells and T cells as well as reduced serum and pulmonary Ig levels, increasing the risk, for example, of pneumonia (Libertucci and Young 2019).The administration of specific commensal bacteria to humans also increases the virus neutralizing serum Igs in response to viral vaccination (de Vrese, Rautenberg, et al. 2005).In the context of COVID-19 infection, the microbiota of the human subject has been associated with the response to vaccination as indicated above (Ng, Peng et al.), suggesting a role in the defense against SARS-CoV-2 infection by priming IgA producing B cells.Approximately 70% of non-severe COVID-19 patients have high and persistent SARS-CoV-2 neutralizing Ig G, A and M in the sera after their recovery.However, while IgG is stable, IgA and M rapidly decrease (Isho, et al. 2020).Anti-SARS-CoV-2 specific IgG and IgA were found also in saliva of COVID-19 patients (Isho, et al. 2020), indicating that antibody in the gastro intestinal tract could be as crucial as antibodies in the serum for protective immunity.Antibiotic treatment impaired CD4 + and CD8 + T cell function in mice, and show reductions in antigen specific IFN-γproducing cytotoxic CD8 T cells against viral infection, increasing mortality rates (Gonzalez-Perez et al. 2016).These findings indicate that the commensal microbiota is critical for sustaining the adaptive anti-viral immune response driven by T effector cells.Additionally, subjects with severe COVID-19 infection present lymphopenia, a reduced number of circulating lymphocytes (Vabret et al. 2020), which theoretically could be linked to gut microbiome alterations that are reported to be especially important in severe cases.Also, SCFA, which are metabolites generated by the intestinal microbiota, are shown to protect against influenza virus infection by enhancing the function of CD8 T cells (Trompette et al. 2018).Although one in vitro study showed that SCFA did not alter the viral load of infected ex vivo human intestinal epithelial cells (Pascoal et al. 2021), the possibility that the SCFA have a beneficial effect on SARS-CoV-2 infection cannot be excluded since in vivo these effects may be dependent on interactions with different cell types (Wlodarczyk, et al. 2022).

Gut-lung immune axis in SARS-CoV-2 infection
Given the exposure to constant external stimuli, the lungs are essential organs in the body's defence mechanisms (He et al. 2017).Several studies have demonstrated the effect of the gut microbiota on lung health through a bidirectional exchange between the gut microbiota and the lungs, involving for example the migration of immune cells from the gut to the respiratory tract to fight infection, which is defined as the gut-lung axis (Keely, Talley, and Hansbro 2012;Dumas et al. 2018).Although, mechanistically, this phenomenon remains poorly defined, the existence of the gut-lung axis and its implications in health and disease could be a key for the prognosis and treatment of several diseases, such as COVID-19.Changes in the gut microbiota composition are associated with an increase in susceptibility to respiratory diseases.For example, the use of antibiotics, which disrupt the microbiota in early life, has been associated with a higher risk of developing asthma (Korpela et al. 2016).Moreover, a lower abundance of Bifidobacterium, Akkermansia, and Faecalibacterium in the human neonatal gut microbiome is linked to an increased risk of childhood atopy (Fujimura et al. 2016).Additionally, it has been shown that respiratory influenza associates with alteration in the gut microbiota, such as a decreased abundance of Lactobacillus and Lactococcus (Wang et al. 2014).Interestingly, the opportunistic bacterium of the upper respiratory tract, Actinomyces viscosus, has been identified in the microbiota of COVID-19 patients, suggesting that transmission of extra-intestinal microbes to the gut is also part of the gut-lung crosstalk (Zuo, Zhan, et al. 2020).A recent study in mice also highlighted the importance of the gut microbiota and derived metabolites, such as SCFAs, and specially acetic acid, on the host's pulmonary defenses against bacterial infections, increasing survival rates during influenza bacterial infection.(Sencio et al. 2020).Similarly, studies in rodents point to a role of orally administered commensal bacteria in reducing inflammatory cell infiltrates and speeding influenza viral clearance through either activation of the recruitment of DC in the lungs or sustaining the IFN activation (Belkacem et al. 2017;Bradley et al. 2019).Moreover, intestinal dysbiosis in COVID-19 patients is associated with an imbalance of the pulmonary microbiota, suggesting that mucous surfaces may be connected, and changes in one site may have consequences on another site (Fan et al. 2020;Zuo, Zhan, et al. 2020).Even though, some of the reported relationships between gut microbiota and lung immunity could be a secondary consequence of the permeation of gut microbial products, for example, through a leaky gut (Hoel et al. 2021), which can then reach the lung and other organs.Indeed, the specific mechanisms through which the gut influences the lung microbes and immunity or vice versa are far from being fully understood and deserve further investigation.

Gut microbiome modulation to reduce risk and attenuate COVID-19
Given the link between gut microbiota alterations and the host's immune response to SARS-CoV-2 infection, gut microbiome modulation strategies have been considered to treat and/or prevent the disease.Evidence of the role of microbiota-directed therapies as adjuvants for treatment are summarized below.However, to date, there are no similar therapies with a demonstrated effect in reducing the risk of COVID-19 infection.Further insights into microbiota composition and functions, including the generation of immunomodulatory metabolites, before and during COVID-19 pathogenesis may also help to identify preventive and therapeutic targets.

Fecal microbiota transplantation
Fecal microbiota transplantation (FMT) is the administration of fecal matter, representing the whole microbiota, from a donor into the intestinal tract of a recipient in order to change the recipient's gut microbiota and confer a health benefit (Gupta, Allen-Vercoe, and Petrof 2016).Previously, FMT has been used successfully to treat Clostridium difficile infection (Liu et al. 2021).Considering that gut microbiome perturbations are associated with COVID-19 severity, FMT has the potential of being a therapeutic strategy to prevent or ameliorate SARS-CoV-2 infection.To the best of our knowledge, no clinical trial has been accomplished on the safety or efficacy of FMT in COVID-19 patients.Bilinski et al. reported of two patients with rapid resolution of COVID-19 symptoms after FMT was used to treat C. difficile infection (Bilinski, et al. 2022).Based on this work, FMT appears to be an efficient strategy to treat patients with both, C. difficile infection and COVID-19.However, due to the lack of large-scale data, FMT cannot be recommended as a therapy against SARS-CoV-2 infection.A clinical trial (FeMToCOVID) involving 300 subjects is currently ongoing and it will be the first large clinical trial using of FMT in COVID-19.Trial results are expected by December 2022.

Prebiotics
Dietary prebiotics are typically non-digestible fibers that act as an energy source for commensal bacteria, stimulating their growth or activity while decreasing the proportion of harmful species (Hutkins et al. 2016).Moreover, during the fermentation of dietary fibers, several bacterial species produce beneficial metabolites, such as SCFA, which serve to maintain colonic mucosal integrity and modulate the immune system (Ratajczak et al. 2019).A murine model has been used to demonstrate that a high-fibre diet was protective against allergic inflammation in the lungs (Trompette et al. 2018).However, while the use of prebiotics might be useful in the prevention or alleviation of SARS-CoV-2 infection, only one recent study has shown that high intake of dietary fibers with diverse physicochemical structures significantly ameliorates severe gastrointestinal symptoms in a patient with post-acute COVID-19 syndrome (Wang et al. 2022).Also, a small open-label study reported that a symbiotic formula, including galactooligosaccharides, xylooligosaccharide, resistant dextrin and Bifidobacterium strains, might contribute to increasing SARS-CoV-2 IgG antibody production and reducing viral loads and inflammatory immune markers in hospitalized COVID-19 patients (Chen and Vitetta 2021).

Probiotics
Probiotics are live microorganisms which when administered in adequate amounts confer health benefits to the host (Reid 2016).Evidence suggests that probiotics or their structural components or metabolic products, mediate immunoregulatory effects by regulating the functions of systemic and mucosal immune cells and intestinal epithelial cells (Yan and Polk 2011).Thus, some probiotic strains showed therapeutic potential for diseases, such as atopic dermatitis, rheumatoid arthritis, and respiratory tract infections among others (Kiousi et al. 2019).Assays in animal models have shown that Lactobacillus gasseri SBT2055 can prevent respiratory syncytial virus infection (Eguchi et al. 2019).Similarly, the release of multiple inflammatory mediators was reduced in human monocytes treated with strains of Lactobacillus paracasei and Lactobacillus plantarum (Schmitter et al. 2018).Additionally, Shibata and colleagues have demonstrated the anti-influenza activity of Lactococcus lactis JCM5805 which directly stimulated pDCs which, in turn, stimulated IFN production and suppressed viral replication and spreading, in a randomized, placebo-controlled, double-blind study with 396 subjects (Shibata et al. 2016).In humans it has been shown that Bifidobacterium lactis HN019 enhances cellular immunity and that Lactobacillus rhamnosus GG and strains of the species Bacillus subtilis, and Enterococcus faecalis significantly improve symptoms in patients with ventilator-associated pneumonia (Gill et al. 2001;Morrow, Kollef, and Casale 2010;Zeng et al. 2016).After initial reports of gut dysbiosis in patients with severe COVID-19, the China National Health Commission recommended the use of probiotics to maintain gut microbial homeostasis and prevent secondary bacterial infections (Gao, Chen, and Fang 2020).Additionally, given the fact that several probiotics, particularly lactic acid bacteria, produce peptides with angiotensin-converting-enzyme inhibitory effect (Ettinger et al. 2014), probiotics were proposed as an adjuvant strategy in the treatment of COVID-19 patients.Data from several randomized, double-blind, placebo-controlled intervention studies, reported that Lactobacillus casei DN114-001, Lactobacillus plantarum DR7, Lactobacillus rhamnosus GG, Lactobacillus gasseri PA 16/8, Bifidobacterium bifidum MF 20/5, Bifidobacterium longum BB536, Leuconostoc mesenteroides 32-77:1, and Pediococcus pentosaceus 5-33:3 helped to prevent respiratory tract infections, suggesting their use as probiotics for lowering the COVID-19 burden (de Vrese, Rautenberg, et al. 2005;Kotzampassi et al. 2006;Namba et al. 2010;Luoto et al. 2014;Baud et al. 2020).However, to date, few published studies have evaluated the use of probiotics for COVID-19 management.Xu and colleagues observed that 62 COVID-19 patients with relatively mild symptoms had received probiotics, but no indication of the used strains was given (Xu, et al. 2020).Currently, a clinical trial (NCT04847349) investigates the effects of a microbiota formula designed for early management of COVID-19 and prevention of severe complications and hospitalization in high-risk patients who suffer from obesity or type-2 diabetes.In a recent publication, a product including four different bacterial strains, three of Lactobacillus plantarum and one of Pedicococcus acidilactici, was reported to improve remission rate, symptom duration and viral load in COVID-19 outpatients.The authors hypothesized that the administered probiotics may act on the gut-lung axis via crosstalk with the host's immune system either by small bacterial metabolites (i.e., SCFA) that can permeate into circulation to prime immune cells in peripheral tissues or by some bacterial cell surface components recognized by antigen-presenting cells, which could result in systemic effects via migration of primed lymphocytes (Gutierrez-Castrellon, et al. 2022).To date, there is no claim approved by authoritative bodies or recommendations from professional health-care associations on probiotic-based treatments for COVID-19, but further research could open up new scenarios.

Conclusion
Evidence suggests a key role of the gut commensal microbes in activating the host immune system locally and systemically helping fight viral infections and specifically COVID-19.Similarly, the gut microbiome of the individual might act as an adjuvant of COVID-19 vaccines.Discovery of the precise gut microbiome signatures of subjects with different susceptibility to the infection and response to COVID-19 vaccines is critical to progress in the identification of vulnerable

Figure 1 .
Figure 1.immune response against sars-Cov-2 infection.left: normal response against Covid-19 infection, with minimal lung inflammation.right: defective immune response against severe Covid-19 infection.the cytokine storm might lead to a severe lung inflammation and, eventually, to organ damages.

Figure 2 .
Figure 2. Mechanism of sars-Cov-2 viral entry into the host organisms.left: aCe2 and tMPrss2 are expressed in the nasal mucosa, lungs and intestine, making them a potential entry sites for the virus.right: once the sars-Cov-2-binding to aCe2 complex encounter tMPrss2, the virus is internalized by the cells.then, viral rna is released into the host cell cytoplasm for replication.

Table 1 .
summary of relevant studies on Covid-19 and gut microbiota in humans.
sd: standard deviation; na: not available; PaCs: conserve as at least one persistent symptom which could not be explained by an alternative diagnosis 4 weeks after clearance of sars-Cov-2; ns: no significant differences were reported.Table 1.(Continued).