Gut microbiota-derived metabolites offer protection against SARS-CoV-2

By Nidhi Saha, BDSAug 5 2022Reviewed by Aimee Molineux

A new study published in the journal Gut Microbes underscored the significance of fiber-fermenting gut bacteria that release short-chain fatty acids (SCFAs), which limit viral entry and hypercoagulation and induce an immune-mediated antiviral response.

Background

The gut microbiome comprises billions of bacterial cells that inhabit the digestive tract. These microbial communities significantly influence the physiology of both health and diseases. The gut microbiome plays a crucial role in the execution of fundamental functions in the body; through these, they impact most pathophysiological processes.

Researchers have unraveled multifaceted functions of the gut microbiome, for instance, its role in the coagulation processes. Gut microbiomes are of particular importance in viral respiratory infections, such as influenza and COVID-19, as well as in chronic lung diseases like asthma and COPD. A favorable, healthy gut microbiome can improve the outcomes of viral infections.

Results of recent investigations depict that COVID-19 patients have a variant gut microbiota. Angiotensin-converting enzyme 2 (ACE2) promotes the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and precipitates coronavirus disease 2019 (COVID-19) by facilitating the production of angiotensin-converting enzyme.

Many cells in the human body express ACE2, including the epithelial cells lining the gastrointestinal (GI) tract and those in the respiratory tract. Up to fifty percent of COVID-19 patients exhibit gastrointestinal symptoms as the earliest signs of SARS-CoV-2 infection.

In convalescent or asymptomatic COVID-19 patients, persistent intestinal reservoirs of SARS-CoV-2 may contribute to the evolution of B cell memory responses. Another characteristic that exacerbates mortality and morbidity associated with COVID-19 is coagulation response dysfunction. However, the relevance of the gut microbiota in SARS-CoV-2 and COVID-19 susceptibility remains unknown.

The study

The purpose of this study was to investigate the mechanisms through which the gut microbiome confers immunity to mammalian hosts against SARS-CoV-2 intranasal infection by generating short-chain fatty acids (SCFAs).

QPCR analysis was conducted on a group of C57BL/6 mice raised in germ-free conditions – either wild-type or K18-hACE2 mice, to compare the ACE2 expression that was specific pathogen-free (SPF) and germ-free (GF). The expression of ACE2 protein in kidney cells of GF mice was assessed by flow cytometry, western blot, and immunofluorescence imaging. SPF mice were administered gentamicin or vancomycin in drinking water for two weeks to identify gut bacteria that modulate the ACE2 expression.

Syrian hamster males, aged nine weeks, were fed SCFA water for two weeks before intranasal infection with a replication-incompetent vesicular stomatitis virus (VSV) pseudovirus, with SARS-CoV-2 spike protein and nanoluciferase.

Additionally, the mice were administered pectin and other dietary fibers to determine the effect of fiber-fermenting commensals. Mice infected with the SARS-CoV-2 Gamma variant were exposed to SCFA water for two weeks before infection, and lung tissue was collected 2.5 days post-infection when the lung viral titers peaked.

SCFA water was administered to hACE2 mice (mice expressing both mouse and human ACE2) for two weeks before intranasal infection with rVSV/Spike-nLuc (a replication-competent chimeric VSV virus with SARS-CoV-2 spike and nanoluciferase) – capable of interacting with human ACE2, but not mouse ACE2. Luciferase activity was assessed in the nasal epithelium, lungs, and small intestine of SCFA-treated mice, compared to the control mice.

The memory response of hACE2 mice was evaluated by administering a primary dose of 6x10⁴ PFU of rVSV/Spike-GFP (SCFA treated GF mice infected with a replication-competent chimeric VSV virus with GFP and the SARS-CoV-2 beta variant spike protein) intranasally, followed by a secondary dosage of 3x10⁵ PFU after four weeks. Blood immune cells were analyzed by flow cytometry two weeks following the first infection.

Ribonucleic acid (RNA) sequencing was performed on the lungs of male GF mice fed either control or SCFA water for two weeks to examine the effect of additional pathways elicited by COVID-19 infection.

The findings

In the gut and lung, Clostridia and SCFAs inhibit ACE2 expression.

Major producers of SCFAs such as acetate, butyrate, and propionate are Clostridia. By producing SCFA, commensal Clostridia species inhibit the expression of ACE2. SCFA treatments inhibited the expression of ACE2, decreased viral loads, and enhanced adaptive immunity against VSV/SARS-CoV-2 chimeras.

SCFAs protect mice against intranasal infection.

A reduction in luciferase activity was observed in the lung epithelium and nasal epithelium of SCFA-treated hamsters compared to control hamsters, indicating a reduced incidence of infection.

In response to intranasal infection with a VSV pseudovirus producing the spike protein, SCFAs enhance T-cell antiviral responses, antibody neutralization, and spike-specific B and T cells. It is noteworthy that these phenotypes were observed in both hACE2 mice, whose expression of human ACE2 did not falter on SCFA treatment, as well as in wild-type mice inoculated with heat-inactivated virus. These findings suggested that the observed enhancement of immune responses is independent of the effect of SCFAs on ACE2-mediated viral entry.

SCFAs improve adaptive immunity to VSV/SARS-CoV-2 chimeric viruses via GPR41/GPR43.

In mice treated with SCFA, there was a trend toward reduced weight loss, as well as a reduction in viral titers and viral RNA in the lungs. In the presence of SARS-CoV-2 or VSV-SARS chimeric viruses, SCFAs lower the viral burden.

Based on flow cytometric analysis, it was found that CD4+ and CD8+ T cells expressed greater levels of interferon (IFN); the regulatory T cells (Treg) formation was enhanced, and granzyme B production was elevated in CD8+ T cells. Dendritic cells were more abundant in the lungs of mice treated with SCFA, but not macrophages or polymorphonuclear cells (PMNs). It was also shown that SCFAs stimulated antiviral immune responses in the lungs during acute infection.

SCFAs regulate the coagulation response via the Sh2b3-Mpl axis to optimize platelet turnover.

A significant negative regulator of megakaryopoiesis and thrombopoiesis, the gene Sh2b3 (also known as lymphocyte adaptor protein – LNK), plays a fundamental role in regulating the coagulation response through its involvement in a variety of signal transduction pathways. SCFAs were shown to promote better SARS-CoV-2 infection outcomes by impeding the coagulation response through the Sh2b3-Mpl axis, according to a third possible axis.

Of note, in the current study, SCFAs influenced ACE2 expression and antibody neutralization in male mice but not the coagulation response. It was found that SCFA-mediated overexpression of Sh2b3 inhibits Mpl (myeloproliferative leukemia protein) signaling in megakaryocytes – resulting in a decrease in megakaryocyte proliferation and platelet production as well as a restriction of platelet turnover.