Microbiota-Immune System Interactions and Enteric Virus Infection

Abstract

Enteric viruses are important human pathogens that pose a significant global health problem. These viruses infect the gastrointestinal tract, which contains a community of microbes called the “microbiota”. We and others have shown that intestinal microbiota are crucial for the replication, pathogenesis, and transmission of a variety of enteric viruses. However, the mechanisms underlying microbiota enhancement of enteric virus infection remain unclear. Interestingly, the host immune system is dependent on both the abundance and composition of the intestinal microbiota. Here we review several aspects of how microbiota influence the immune system and how this could potentially impact enteric virus infection.

Introduction

Within the gastrointestinal tract resides a microbial ecosystem of approximately 10¹⁴ organisms, which play a crucial role in host homeostasis (1). Importantly, microbiota are critical for the development of the host immune system (2). Specifically, microbiota influence lymphoid structure development, immune cell homeostasis and maturation, epithelial function, T cell development, induction of diverse immune cascades, production of antimicrobial peptides, inflammation, and autoimmune diseases (1, 2). Additionally, microbiota affect host biosynthesis of vitamins, hormones, and neurotransmitters, and production of bacterial byproducts such as short-chain fatty acids (SCFAs) (1, 2). Conversely, microbiota are shaped by the host environment, diet, and the use of antibiotics (3). Dysbiosis of the microbiota has been linked to a variety of diseases, including gastrointestinal, neurologic, metabolic, cardiovascular, and oncologic disease (1).

Enteric viruses are acquired through the fecal-oral route of transmission, where they infect the gastrointestinal tract, and directly contact the microbiota (4). This interaction with the microbiota has a variety of effects on enteric viruses (4). However, little is known about the interplay between microbiota, the host immune system, and enteric viruses. In this review, we discuss how the microbiota influence host homeostasis and intestinal immunity, and how these processes potentially influence enteric virus infection.

Microbiota priming of host immunity

Millions of years of coevolution between host and microbiota has forged a symbiotic relationship (2). Models of microbiota manipulation, such as germ-free or antibiotic treated mice, demonstrate altered immune response of the host (3, 5). Specifically, germ-free mice, which completely lack microorganisms, have altered immune cell homeostasis, impaired lymphoid tissue development, and altered susceptibility to pathogens, suggesting that microorganisms are crucial for proper host immunity (3). Intriguingly, little is known about how these changes impact enteric virus replication and whether the mechanisms are conserved or divergent.

Both antibiotic-treated and germ-free mice have decreased myeloid cell populations, which are known to impact a variety of viral infections (3, 6). Interestingly, macrophages are decreased in the small intestine and colon upon antibiotic treatment, and these tissues are important sites of replication for a variety of enteric viruses (7•,8•–10). Additionally, antibiotic-treated mice have impaired basal type I interferon production, and reduced populations of dendritic cells (DCs), CD4+ T cells, CD8+ T cells, T regulatory cells (FoxP3+), Th1 cells (IFNγ+), and B cells in the small intestine and colon (11). We and others have shown that several enteric viruses have decreased viral replication in intestinal tissues in antibiotic-treated mice (7•,8•,9,12). However, the mechanism for this reduction in viral replication remains unknown and it is unclear how these diverse cell populations influence enteric virus infection. To address this question, it will be important to determine how reducing specific cell populations influences enteric virus infection.

Another aspect of the immune system that is modulated by the microbiota is the production of cytokines, which have varying effects on viral infection (3, 11). Interestingly, germ-free or antibiotic-treated mice have altered cytokine levels in a variety of tissues. Specifically, several cytokines important for enteric virus infection are reduced in intestinal tissue of microbiota-depleted mice, including IL-1β, TNF-α, and IL17 (13–15). Overall, alteration of the microbiota greatly influences cytokine levels in both gastrointestinal and systemic tissues. Future studies could focus on how changes in cytokine levels in gastrointestinal tissues impact enteric virus infection.

To complicate matters, antibiotics have microbiota-independent effects on both host immunity and viral infection, meaning that antibiotics can directly modulate host cells (16•, 17•). This makes it difficult to discern whether immune changes are due to the absence of bacteria or direct effects of antibiotics on host cells. To further our understanding of the effects of antibiotics, it is crucial determine whether antibiotic effects are microbiota-dependent vs. microbiota-independent by treating germ-free mice with antibiotics and monitoring consequences.

Immune modulation by bacterial derived short-chain fatty acids

A gap in the field of microbiota-virus interactions is how bacteria can indirectly influence the immune system through production of SCFAs. Bacteria within the gastrointestinal tract ferment dietary fiber to produce SCFAs such as butyrate, propionate, and acetate (18). Through fermentation of dietary fiber, bacteria produce millimolar quantities of SCFAs, making them some of the most abundant molecules in the gut (19). SCFAs are primarily produced by anaerobic bacteria, most notably Gram-positive Firmicutes in the class Clostridia, although other bacteria can produce SCFAs as well (20).

SCFAs influence host immunity by a variety of mechanisms, such as altering gene expression and intestinal metabolism, which in turn could potentially impact enteric virus infection (21–23). Additionally, SCFAs can alter immune cell development and function, such as altering regulatory T cell homeostasis in the gut (22). Interestingly, when mice were given a single dose of streptomycin, they exhibited a marked decrease in cecal Clostridia and butyrate (24). Importantly, this single dose of streptomycin also reduced shedding and pathogenesis of the enteric virus coxsackievirus B3, but not poliovirus, suggesting that two closely related enteric viruses have different requirements of the host microbiota (8).

One SCFA, butyrate, influences intestinal immunity through changes in host gene expression in the gut. Butyrate alters gene expression through inhibition of histone deacetylases, and by creating a hypoxic state through beta-oxidation of butyrate, which stabilizes the transcription factor hypoxia inducible factor 1 (25, 26). Furthermore, the intestinal inflammatory response is altered by butyrate through activation of G-protein coupled receptors (20, 27, 28). Some of the genes affected by butyrate influence viral infection. For example, butyrate upregulates expression of the coxsackievirus and adenovirus receptor (CAR), which is the receptor for coxsackievirus B3 (29). Butyrate also downregulates various innate immune genes such as CCL2, CCL7, CXCL2, IL6, IL12, NOS2, and TNF-α (21, 23, 30). A recent study demonstrated that, in cultured cells, butyrate treatment increased infection with several viruses by suppressing expression of interferon-stimulated genes (31•). Taken together, bacterial-derived butyrate regulates various innate immune genes that are important for enteric virus infection.

These findings suggest that butyrate, and perhaps other SCFAs, may influence enteric virus infection in the intestine (Figure 1). Future studies could examine the impact of specific SCFAs on a variety of enteric viruses using mouse models. For example, depletion of microbiota with antibiotics, followed by treatment with exogenous acetate, butyrate, or propionate prior to enteric virus infection could determine whether SCFAs are sufficient for enteric virus replication. Furthermore, it remains unknown if and how SCFA-mediated changes to host gene expression affects enteric virus infection. Unbiased approaches, such as analysis of SCFA-mediated transcriptomic changes by RNA sequencing in different tissues throughout the gastrointestinal tract (from conventional, antibiotic treated, or antibiotic treated plus specific SCFA mice) could reveal shared vs. unique gene expression signatures relevant to enteric virus infection.

Fig 1. Influence of Intestinal Microbiota on Host Physiology.

Microbially-derived short-chain fatty acids alter innate immune genes relevant to enteric virus infection. Both microbiota and microbial-derived metabolites are influenced by circadian rhythms. Upon antibiotic treatment, short-chain fatty acids are significantly reduced. Antibiotic treatment also reduces enteric virus infection. It remains unknown if exogenously added metabolites are sufficient to rescue enteric virus infection. Figure was created with BioRender.

The microbiota also modifies host metabolites such as bile acids, which can have both pro- and anti-viral effects on certain enteric viruses. Specifically, these bile acids bind to the human norovirus capsid protein and are important cofactors for efficient binding to CD300lf, the cell surface receptor for murine norovirus (32, 33). Interestingly, bacterial-modified bile acids prime type-III interferon induction to suppress murine norovirus infection in the proximal intestine (34). Furthermore, bile acids suppress rotavirus replication by downregulating rotavirus-induced lipid synthesis, suggesting that bile acids influence the replication of enteric viruses through various mechanisms (35). Taken together, bacterial-modified bile acids modulate infection of various enteric viruses.

Circadian effects on microbiota, immunity, and enteric virus infection

There has been growing evidence of a link between circadian rhythms and oscillations in the microbiota and host response pathways. Circadian rhythms are roughly 24-hour oscillations in a variety of processes that are entrained by the diurnal cycle of the planet (36). This rhythmicity is perpetuated by a set of transcription factors, so called “clock proteins”, which control expression of ~3–20% of genes in any mammalian cell or tissue. Thus, major physiological processes are under circadian control, including metabolism and immunity (37, 38). Additionally, microbiota undergo diurnal oscillations in both function and composition along the different tissues of the gastrointestinal tract (35•, 36•). These fluctuations in the microbiota are driven by host feeding rhythms (41). Furthermore, fluctuation in the amount of bacterial attachment to the colonic epithelium is also driven by diurnal oscillations (35•, 36•). However, little is known about how these fluctuations impact immune cell populations throughout the gastrointestinal tract. Moreover, it remains unclear if cytokine levels fluctuate throughout the gastrointestinal tract over a 24-hour circadian cycle. To date, it remains unknown how circadian rhythms impact enteric virus infection. Since enteric viruses require the host microbiota to establish infection in the host, it will be illuminating to determine the impact of microbial diurnal oscillations on intestinal immunity and how this influences enteric virus infection.

Interestingly, circadian disorders can cause microbial dysbiosis, which impacts host metabolism (35•). Feces isolated from mice displayed significant rhythmicity in the levels of SCFAs such as acetate, propionate, and butyrate, which are important molecules for driving host metabolism and immunity (42). Furthermore, intestinal microbiota are required for rhythmic recruitment of histone deacetylase 3 to target gene promoters, which rhythmically regulates host metabolism and drives expression of hundreds of downstream gene targets (36). Given the importance of SCFAs in modulating host immunity and that SCFAs are known to fluctuate throughout the day, it would be interesting to determine whether enteric virus infection is dependent on the time of day that the host is inoculated.

Conclusions

Considering all these findings, there is a clear need to further understand how the intestinal microbiota influences enteric virus infection and whether microbiota derived effects on host immunity is playing a role in enteric virus infection. Many questions remain about mechanisms of both direct and indirect effects on the microbiota on enteric virus infection. Which immune cells influence enteric virus infection and are these populations altered by the microbiota? Do changes in host immunity through abundance of microbial-derived SCFAs impact enteric virus enteric virus infection and if so, how? What is the impact of circadian rhythms on enteric virus infection and do microbiota fluctuations play a role? Elucidating the effects of the microbiota on the immune system and host homeostasis and how this influences enteric virus infection is a promising field of study. Future studies on the host microbiota, and how they modulate host immunity, may inspire new therapeutic approaches targeting enteric viruses.