Microbiota-Nourishing Immunity: A Guide to Understanding Our Microbial Self

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Review Microbiota-Nourishing Immunity: A Guide to Understanding Our Microbial Self €umler1,* Yael Litvak1 and Andreas J. Ba 1Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, CA 95616, USA *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2019.08.003

In ecological terms, the microbiome is defined as the microbiota and its environment, a definition that encompasses the human host. The size, species composition, and biogeography of microbial communities is shaped by host interactions, and, in turn, the microbiota influences many aspects of human health. Here we discuss the concept of microbiota-nourishing immunity, a host-microbe chimera composed of the microbiota and host factors that shape the microbial ecosystem, which functions in conferring colonization resistance against pathogens. We propose that dysbiosis is a biomarker of a weakening in microbiota-nourishing immunity and that homeostasis can be defined as a state of immune competence. Microbiota-nourishing immunity thus provides a conceptual framework to further examine the mechanisms that preserve a healthy microbiome and the drivers and consequences of dysbiosis. Introduction Next-generation DNA sequencing has revolutionized the study of microbial communities by establishing culture-independent methods for profiling their diversity ((HMP) Human Microbiome Project Consortium, 2012). This pioneering work revealed that balanced host-associated microbial communities—the microbiota—are linked to health, while an imbalance in the microbiota is associated with a broad range of non-communicable human diseases (Cani, 2017). There is an expectation that the vast amount of information generated by high-throughput technology along with supercomputing can move research on the microbiome beyond a descriptive stage, because with enough data, hypotheses will eventually emerge (Mazzocchi, 2015; Tripathi et al., 2018). This prospect has driven an ever-increasing sophistication of microbiome measurements, with shotgun metagenomics replacing 16S ribosomal RNA gene amplicon sequencing, meta-transcriptomics replacing metagenomics, and single-cell transcriptomics replacing community-based sequencing. But despite these extraordinary advances in data collection technologies, we are still no closer to specifying what constitutes a healthy microbiome (Proctor, 2019). Terms such as dysbiosis and homeostasis remain poorly defined (Olesen and Alm, 2016) despite an avalanche of data being generated through an ever-increasing depth of microbiota measurements. It is becoming increasingly clear that the speed at which technology-based empiricism translates vast amounts of data into theory is slow. Perhaps it is time to explore whether complementing this approach by injecting a healthy dose of deductive reasoning could accelerate the generation of an overarching conceptual framework for understanding how the microbiota is balanced during homeostasis and how this balance is disrupted during dysbiosis. The microbiome is commonly described as the microbiota, its genes, and gene products, a definition that puts the focus entirely on the microbes (Hooper and Gordon, 2001). However, in ecological terms, the microbiome is defined as the microbiota and its environment (Tipton et al., 2019), a definition that encompasses the human host, who plays a major role in shaping the 214 Immunity 51, August 20, 2019 ª 2019 Elsevier Inc.

environment inhabited by the microbiota (Byndloss et al., 2018; Foster et al., 2017). Consequently, host-derived factors that shape the microbial ecosystem contribute to a healthy microbiome just as the microbiota and its metabolites do. An overarching framework for microbiome research thus needs to incorporate host-derived ecological forces as well as the microbiota and its metabolic activity. The concept that the host immune system evolved to shape the microbiota to be beneficial (Foster et al., 2017) links host-derived ecological forces to the composition and function of the microbiota (Box 1). However, contemporary microbiome research has made it increasingly clear that interactions between our immune system and the microbiota represent a type of immune response that is distinct from classical anti-infective host responses that bestow sterilizing immunity. Immune responses mediating sterilizing immunity function in detecting microbes that enter our tissue and in eliciting responses to remove them from our body. In contrast, the goal of interactions between our immune system and the microbiota is to maintain and shape microbial communities on host surfaces, an objective that is fundamentally different from sterilizing host tissue. Whereas a century of research has provided a detailed picture of functions involved in sterilizing immunity, mechanisms involved in balancing our microbial communities remain understudied. In this Perspective, we propose a theoretical framework for homeostasis and dysbiosis by incorporating ecological and immunological concepts into microbiome research. This viewpoint suggests that understanding the mechanisms by which the host immune system shapes its microbiota will be an important future goal, as cataloguing microbial genes and species names alone does not reveal what constitutes a healthy microbiome. A Host-Microbe Chimera Forms the Body’s First Line of Defense The germ theory had a foundational influence on concepts of sterilizing immunity by establishing microbial pathogens as the cause of communicable diseases, which in turn inspired work

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Review Box 1. The Host Shapes Its Microbiota to Be Beneficial

The idea that the host shapes the microbiota to be beneficial originates from ecological paradigms on how invertebrate hosts sculpt microbial communities to their advantage (Foster et al., 2017). One striking example is the light organ of the bobtail squid (Ruby, 1996). The squid shapes the environment within its light organ to restrict access to a single bacterial species, the luminous Vibrio fischeri. Species-specific selection mechanisms that shape the bacterial community include physical defenses provided by a mucociliary epithelial surface on the light organ’s exterior (Altura et al., 2013), epithelial release of antimicrobial peptides that suppress growth of other bacterial species (Troll et al., 2010), nourishment of V. fischeri through host endochitinase-mediated mucus hydrolysis (Kremer et al., 2013), and host-derived bactericidal phenoloxidase activity in the light organ (Kremer et al., 2014). Light production by luciferase is under positive selection, because the bioluminescence reaction consumes oxygen, thereby protecting V. fischeri from phenoloxidase activity (Kremer et al., 2014). In turn, light production enables the bobtail squid to match its background, thereby camouflaging itself from predators and prey. This example illustrates that shaping the microbiota provides benefit to the host, which is not limited to mediating colonization resistance or aiding in food digestion.

on mechanisms by which our immune system detects and eliminates these intruders from host tissue. However, to understand a healthy microbiome, it is more useful to think of the microbiota as an organ-like collection of microbes that provides benefit to the host (Marchesi et al., 2016; O’Hara and Shanahan, 2006). The idea that the host immune system maintains functionality of our microbial organ during homeostasis is known as the germ-organ theory (Byndloss et al., 2018). An important health benefit provided by our microbial organ is its ability to confer niche protection against pathogens, a core function of the microbiota called colonization resistance (Libertucci and Young, 2019; €umler, 2019). Immune processes that shape and Litvak and Ba maintain the microbial ecosystem thus form a functional unit together with our microbial organ, and we recently proposed the term microbiota-nourishing immunity to describe this hostmicrobe chimera (Byndloss et al., 2019) (Figure 1A). Whereas sterilizing immunity removes microbial intruders from tissue solely through host-derived antimicrobial immune mechanisms, microbiota-nourishing immunity repels pathogens from host surfaces using a microbial organ. Although it contains a microbial component, microbiota-nourishing immunity is an integral part of our immune system, because colonization resistance prevents harmful microorganisms from entering the body, which represents a canonical nonspecific immune function. The host components of microbiota-nourishing immunity ensure that microbial communities remain advantageous by limiting their size, sculpting their spatial organization, or shaping their composition (Figure 1B). Functions that limit the size of microbial communities include physical defenses (e.g., the mucus escalator in the respiratory tract [Quie, 1986] or peristalsis in the small intestine [Kongara and Soffer, 2000]), secretions (e.g., gastric acid [Sarker et al., 2017] or pancreatic secretions [Trespi and Ferrieri, 1999]), or immunoglobulin (Pignata et al., 1990). For instance, microbial communities in the small intestine rapidly consume simple carbohydrates, thereby interfering with nutrient absorption (Zoetendal et al., 2012). Furthermore, excessive deconjugation of bile acids by bacteria during small intestinal bacterial overgrowth results in malabsorption of fat and liposoluble vitamins (Fan and Sellin, 2009). Thus, by preventing small intestinal bacterial overgrowth using gastric acid, peristalsis, pancreatic secretions, and immunoglobulin, the host shapes the microbiota to limit potential adverse effects, such as weight loss, malnutrition, and malabsorption (Bures et al., 2010), while maintaining

a community that confers benefit by mediating colonization resistance. Immune functions sculpting the biogeography of microbial communities include the generation of chemical gradients or the release of antimicrobial peptides and proteins. One example is the epithelial release of a bactericidal C-type lectin (RegIIIg), which is thought to restrict numbers of surface-associated Gram-positive bacteria, thereby maintaining a zone that physically separates the microbiota from the epithelium to protect the mucosal surface in the intestine (Vaishnava et al., 2011). Another example is human defensin 5 (HD5), an antimicrobial peptide released from small intestinal Paneth cells, which excludes Gram-positive bacteria from the microbiota that are intimately attached to the epithelial surface, such as segmented filamentous bacteria (phylum Firmicutes) (Salzman et al., 2010). Antimicrobial peptides and proteins are also employed by sterilizing immunity, which illustrates that there is some overlap between the immune mechanisms used to clear bacteria from tissue and those that shape the biogeography of microbial communities on body surfaces. Beyond functions associated with immune effectors, host functions can shape the microbiota composition by controlling the availability of growth-limiting resources. This can be accomplished through mechanisms that literally nourish the microbiota, a feature that distinguishes microbiota-nourishing immunity from sterilizing immunity, which does not employ this mechanism. Processes that nourish the microbiota include milk oligosaccharides, a maternal host control mechanism that maintains a dominance of Bifidobacterium infantis (phylum Actinobacteria) in the infant gut microbiota (Sela et al., 2008); mucus secretion, which sustains colonic populations of Bacteroides species (phylum Bacteroidetes) during periods of fiber starvation (Sonnenburg et al., 2016; Sonnenburg et al., 2005) or by the breakdown of host-derived glycogen into maltose; and maltotriose, which drives a dominance of Lactobacillus species (phylum Firmicutes) in the female reproductive tract (Spear et al., 2014). Alternatively, the microbiota composition can be shaped by withholding growth-limiting resources. An example of the latter mechanism is epithelial hypoxia in the large intestine, which limits oxygen availability in the lumen, thereby constraining growth of facultative anaerobic bacteria that can use this resource to outgrow obligate anaerobic bacteria (Byndloss et al., 2017). The microbial component of microbiota-nourishing immunity confers colonization resistance against pathogens through Immunity 51, August 20, 2019 215

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Review Figure 1. Principles of MicrobiotaNourishing Immunity (A) Sterilizing immunity and microbiota-nourishing immunity form two distinct branches of the immune system. Whereas sterilizing immunity is dedicated to detecting microbes and removing them from host tissue, microbiota-nourishing immunity functions in maintaining a germ organ that repels pathogens from host surfaces. (B) The microbiota and its environment are two components of the microbiome. Microbiota-nourishing immunity, the branch of our immune system that confers colonization resistance against pathogens, is a host-microbe chimera composed of the microbiota and host factors that shape the microbial environment.

mechanisms that are grounded in microbial ecology (Litvak and €umler, 2019). The microbiota is assembled after birth by a Ba stochastic incorporation of maternal or environmental microorganisms (Sprockett et al., 2018) that can coexist because each consumes a few of the available nutrients better than any other member within the microbial community, a concept known as the nutrient niche hypothesis (Freter et al., 1983). Once available nutrient niches are filled by suitable occupants, the microbiota reaches a stable equilibrium state (Sommer et al., 2017) and the microbiota composition becomes resistant to change because founding members of a nutrient niche gain a competitive advantage over newly arriving microbes through niche preemption or niche modification (Fukami, 2015; Sprockett et al., 2018). The priority effects resulting from niche preemption or niche modification are an important determinant of colonization resistance (Litvak et al., 2019; Vannette and Fukami, 2014) (Figure 1B). When a pathogen arrives at a mucosal surface, it faces the problem that due to niche preemption the best seats in the house are already taken and priority effects make it difficult to remove resident microbes to free up a nutrient niche suitable €umler, 2019). Through this mechfor the pathogen (Litvak and Ba anism, microbiota-nourishing immunity can confer colonization resistance by preventing pathogens from gaining access to growth-limiting resources, thereby driving the intruders to extinction (Deriu et al., 2013; Litvak et al., 2019; Velazquez et al., 2019). Alternatively, occupants of a nutrient niche can confer colonization resistance through niche modification. For example, some members of the gut microbiota, such as Clostridium scindens (phylum Firmicutes), convert primary into secondary bile acids (Wells and Hylemon, 2000). The latter limit growth of Clostridioides difficile (phylum Firmicutes), thereby conferring colonization resistance against this opportunistic pathogen through niche modification (Buffie et al., 2015; Theriot et al., 2014). In summary, there is some overlap in antimicrobial mechanisms employed by sterilizing immunity and microbiota-nourishing immunity. However, whereas antimicrobial mechanisms of sterilizing immunity serve as effector functions that aid in clearance of microbes from tissue, microbiota-nourishing immunity uses these host responses to maintain functionality of its microbial component. In turn, this microbial organ confers colonization resistance, which represents the antimicrobial effector function of microbiota-nourishing immunity. Thus, antimicrobial effector functions of sterilizing immunity are host derived, 216 Immunity 51, August 20, 2019

whereas microbiota-nourishing immunity employs microbederived antimicrobial effector functions. However, to understand homeostasis, it is necessary to incorporate microbiota-mediated functions into the picture that go beyond colonization resistance. Germ-Organ Accessory Functions and Homeostasis By encompassing immune functions that shape the microbial ecosystem as well as microbial ecology and microbiota-derived metabolites, microbiota-nourishing immunity transcends all aspects of microbiome research (Figure 1B). These properties make microbiota-nourishing immunity an attractive candidate for an overarching conceptual framework to explain how the host maintains a healthy microbiome. Whereas colonization resistance is a core function shared between body surfaces, microbial communities may perform accessory functions that are specific to one body habitat. Maintaining colonization resistance is thus not sufficient for sustaining homeostasis, because accessory functions need to be preserved as well to ensure that the microbiota remains beneficial. Each body habitat in a healthy human microbiome harbors a distinctive microbial community ((HMP) Human Microbiome Project Consortium, 2012), but knowledge about the accessory functions executed by each of them remains incomplete for most body sites. One notable exception is the colon, which harbors a large microbial community that performs an important accessory digestive function by breaking down nutrients, such as complex carbohydrates (fiber), that cannot be digested by host enzymes in the upper gastrointestinal tract. Knowledge about its accessory function makes the colonic microbiota a good candidate for testing the utility of the microbiota-nourishing immunity concept for illuminating which features characterize a healthy microbiome. To support a large and diverse microbial community that can satisfy our digestive needs, host functions that limit population size are muted in the large intestine, which explains the steep increase in the bacterial density as contents move from the ileum (104–107 bacteria/mL) into the colon (1011–1012 bacteria/mL) €ckhed, 2016). The host spatially separates (Sommer and Ba this large microbial community from the mucosal surface by maintaining an inner mucus layer above the epithelial cells that is mostly devoid of bacteria (Johansson et al., 2008). The accessory function of the colonic microbiota contributes to host nutrition (Vela´zquez et al., 1997) and immune education (Arpaia et al., 2013; Atarashi et al., 2011; Furusawa et al., 2013; Smith et al., 2013) by converting fiber anaerobically into fermentation

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Review products, such as short-chain fatty acids, which are absorbed by the host (den Besten et al., 2013). To preserve this beneficial digestive task, the host polarizes the energy metabolism of the epithelial surface in the colon toward high mitochondrial oxygen consumption, which maintains the epithelium in a state of physiological hypoxia (<1% oxygen) (Furuta et al., 2001). Epithelial hypoxia limits the luminal availability of a growth-limiting resource, oxygen (Byndloss et al., 2017), and the consequent anaerobiosis shapes the microbiota to be dominated by obligate anaerobic bacteria belonging to the classes Clostridia (phylum Firmicutes) and Bacteroidia (phylum Bacteroidetes) ((HMP) Human Microbiome Project Consortium, 2012), two taxa that encode a broad spectrum of enzymes involved in fiber degradation (El Kaoutari et al., 2013). Although changes in dietary fiber consumption can shift the microbiota composition (Shepherd et al., 2018; Sonnenburg et al., 2016), one could argue that this variation remains within the normal range as long as a dominance of obligate anaerobic bacteria is maintained within the colonic microbiota. The emerging picture suggests that a healthy microbiome in the colon is maintained by keeping bacteria physically separated from the epithelial surface and by preserving anaerobiosis in the lumen to support a large and diverse community of obligate anaerobic bacteria. In turn, this microbial community not only breaks down a broad range of complex carbohydrates into fermentation products that are absorbed by the host but also confers colonization resistance, effectively maintaining homeostasis. Thus, the framework of microbiota-nourishing immunity provides a functional definition of gut homeostasis. Identification of additional microbiota accessory functions will help to further refine this concept and make it applicable to other body surfaces. Causes and Consequences of Dysbiosis The term dysbiosis is often used to describe a difference in the microbiota composition between healthy individuals and those with disease, but this definition is ambiguous in that this association between health status and microbiota composition does not distinguish between dysbiosis being the cause or the result of disease (Olesen and Alm, 2016). The idea that microbiotanourishing immunity maintains a healthy microbiome implies that dysbiosis is secondary to a weakening of immune mechanisms that maintain homeostasis. To examine this concept, we will first consider how weakening the host components of microbiota-nourishing immunity affects the microbiota composition in the large intestine, where we have the most comprehensive picture of how a healthy microbiome is maintained. Dysbiosis in the large intestine is characterized by a shift in the microbial community from obligate to facultative anaerobic bacteria, which often includes an increased abundance of members belonging to the phylum Proteobacteria (Shin et al., 2015). The oxygen hypothesis proposes that the increased abundance of Proteobacteria in the colon during dysbiosis is due to a disruption in anaerobiosis (Rigottier-Gois, 2013). One mechanism that disrupts anaerobiosis in the colon is increased epithelial oxygenation, which was first described in studies on intestinal inflammation triggered by enteric pathogens (Lopez et al., 2016; Rivera-Cha´vez et al., 2016). A second factor driving an expansion of Enterobacteriaceae (phylum Proteobacteria) in the colon is epithelial-derived

nitrate, which can be used by Enterobacteriaceae to promote their growth through anaerobic nitrate respiration (Byndloss et al., 2017). Both oxygen and nitrate contribute to an expansion of commensal E. coli (family Enterobacteriaceae) in mouse models of chemically or genetically induced colitis (Hughes et al., 2017; Winter et al., 2013). A dysbiotic expansion of Proteobacteria could thus be considered a microbial signature of a dysfunctional epithelium that no longer limits the availability of growth-limiting resources, such as oxygen and nitrate (Litvak et al., 2017). In case of severe intestinal inflammation, phagocytes can enter the intestinal lumen and consume oxygen by undergoing a respiratory burst, thereby turning the mucosal surface hypoxic (Campbell et al., 2014; Karhausen et al., 2004). However, the respiratory burst of phagocytes generates hostderived electron acceptors, such as tetrathionate and nitrate, which support growth of Enterobacteriaceae through anaerobic respiration (Kamdar et al., 2016; McLaughlin et al., 2019; Winter et al., 2010). Thus, a dysbiotic expansion of facultative anaerobic Proteobacteria in the large intestine is due to an increased availability of host-derived respiratory electron acceptors. The availability of these growth-limiting resources is elevated either because increased epithelial release of oxygen disrupts anaerobiosis or because inflammatory responses increase the availability of nitrate or tetrathionate in the intestinal lumen. Notably, even in cases where dysbiosis is secondary to an underlying host defect, the resulting changes in microbiota composition might be of consequence, because they are often associated with an exacerbation of disease. One example is colorectal cancer, a major cause of cancer-related death. Only about 20% of colorectal cancer cases can be genetically attributed to familial history (Rustgi, 2007), suggesting that environmental factors play an important role in promoting tumorigenesis. One prime candidate for environmental factors is an altered colonic microbiota (Sears and Garrett, 2014). Colibactin-producing E. coli of phylogroup B2 are minority species within a healthy microbiome in a fraction of individuals (Putze et al., 2009). A dysbiotic expansion of colibactin-producing E. coli during colitis can accelerate colorectal cancer formation in a mouse model (Arthur et al., 2012), which makes colibactin-producing E. coli a likely candidate for a pro-oncogenic driver species. Clinical associations link colibactin-producing E. coli to a fraction of colorectal cancer cases (Arthur et al., 2012; Buc et al., 2013). The bacterial driverpassenger model for colorectal cancer proposes that bacterial drivers are gradually outcompeted by other gut commensals (Sears and Pardoll, 2011; Tjalsma et al., 2012). This model thus suggests that clinical associations based on sampling bacteria associated with already-formed tumors might underestimate the importance of driver species. Consistent with an association of colibactin-producing E. coli with stages that precede tumor formation, risk factors for colorectal cancer formation promote an expansion of facultative anaerobic Proteobacteria in the colon, which includes inflammatory bowel disease (Morgan et al., 2012), high-fat diet (Devkota et al., 2012; Martinez-Medina et al., 2014), antibiotic treatment (Byndloss et al., 2017), and alcohol dependence (Dubinkina et al., 2017). In other words, many risk factors for colorectal cancer formation have in common that they generate an environment that favors an expansion of colibactin-producing E. coli. Taken together, these findings suggest that a weakening of microbiota-nourishing immunity Immunity 51, August 20, 2019 217

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Review Figure 2. The PPAR-g Signaling Pathway Controls Mitochondrial Activity to Maintain Homeostatic Microbial Communities in the Large Intestine (A) During homeostasis, the PPAR-g signaling pathway activates mitochondrial activity in the mucosal surface of the colon to maintain the epithelium in a state of physiological hypoxia, thereby limiting an expansion of facultative anaerobic bacteria, such as members of the phylum Proteobacteria. Mesalazine is a drug that can be used to activate the PPAR-g signaling pathway in the colonic epithelium. (B) During dysbiosis, silencing of the PPAR-g signaling pathway reduces mitochondrial activity, thereby increasing epithelial oxygenation, which in turn drives an expansion of facultative anaerobic Proteobacteria. Metformin is a drug that inhibits SIRT3 expression. PGC-1a, peroxisome proliferator-activated receptor-gamma coactivator-1 alpha; PPAR-g, peroxisome proliferator-activated receptor-gamma; SIRT3, Sirtuin 3; TCA, tricarboxylic acid; O2, oxygen.

during inflammatory bowel disease, antibiotic treatment, alcohol consumption, or high-fat diet changes the microbiome to favor a growth of Proteobacteria in the large intestine. In individuals that carry strains of colibactin-producing E. coli, this dysbiosis might escalate the cancer-inducing activity of the microbiota by triggering the bloom of a pro-oncogenic driver species (Arthur et al., 2012), which raises the possibility that impaired microbiota-nourishing immunity in the colon is a potential risk factor for colorectal cancer formation. Microbiota-nourishing immunity can also become compromised through a direct disruption of its microbial components, which is a common side effect of antibiotic therapy. Antibiotic treatment impairs colonization resistance because clearing resident occupants from their respective nutrient niches weakens or eliminates the ability of our microbial organ to repel pathogens through niche preemption or niche modification (Litvak and €umler, 2019). As a result, antibiotic treatment is associated Ba with opportunistic infections caused by C. difficile (Kuijper et al., 2006) or carbapenem-resistant Enterobacteriaceae (phylum Proteobacteria) (Daikos et al., 2010; Lowe et al., 2013; Razazi et al., 2012; Shanthi and Sekar, 2010; Zhao et al., 2016), which currently are classified by the Centers for Disease Control and Prevention (CDC) as two of the most urgent threats to public health (Centers for Disease Control and Prevention, 2014). Although antibiotic treatment directly disrupts our microbial organ, the host is not a passive bystander during antibioticinduced dysbiosis, because some of the changes in the microbiota composition are triggered indirectly by weakening host components of microbiota-nourishing immunity. Microbiotaderived short-chain fatty acids help maintain epithelial hypoxia 218 Immunity 51, August 20, 2019

by enhancing mitochondrial activity through stimulation of PPARg (peroxisome proliferator-activated receptor gamma) signaling (Figure 2A)(Byndloss et al., 2017) and maintenance of regulatory T cells in the colonic mucosa (Atarashi et al., 2011; Smith et al., 2013). By depleting short-chain fatty-acid-producing bacteria, antibiotic treatment depletes short-chain fatty acids (Smith et al., 2013). In turn, short-chain fatty acid depletion reduces mitochondrial activity in the colonic epithelium, and the consequent shift in epithelial metabolism drives a dysbiotic expansion of facultative anaerobic Enterobacteriaceae in the intestinal lumen by increasing the availability of epithelial-derived respiratory electron acceptors, such as oxygen and nitrate (Byndloss et al., 2017; Spees et al., 2013). In conclusion, dysbiosis should be considered a biomarker for a weakening in microbiota-nourishing immunity. Such a biomarker is relevant because it is indicative of an immunodeficiency that can adversely affect human health by either increasing susceptibility to opportunistic infection or triggering changes in the microbiome that exacerbate disease. Implications for Dysbiosis Therapy Most current efforts to treat dysbiosis are targeting the microbiota composition directly, either through fecal microbiota transplantation, probiotics, prebiotics, antibiotics, or precision editing of the microbiota. Fecal microbiota transplantation has proven effective for treating opportunistic C. difficile infections that are associated with an antibiotic-mediated disruption of our microbial organ, which weakens colonization resistance. After cessation of antibiotic therapy, colonization resistance can be reinstated by reestablishing a balanced microbiota through fecal microbiota transplantation in patients (Bakken et al., 2011), which can cure the opportunistic infection by eliminating its underlying cause. Specifically, an antibiotic-mediated depletion of secondary bile acid producers weakens colonization resistance against C. difficile by impairing niche modification (Buffie et al., 2015;

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Review Theriot et al., 2014), but secondary bile acid levels can be restored in patients through fecal microbiota transplantation (Weingarden et al., 2014). A more refined approach for restoring colonization resistance against C. difficile infection in mouse models of antibiotic treatment is a precision microbiome reconstitution with second-generation probiotics, such as C. scindens, that restore niche modification by producing secondary bile acids (Buffie et al., 2015). In mouse models of chemically or genetically induced colitis, dysbiosis can be remediated by selectively blunting an expansion of Enterobacteriaceae. This precision editing of the microbiota is accomplished by inhibiting microbial respiratory pathways using tungstate treatment, which ameliorates the severity of colitis (Zhu et al., 2018), presumably because an expansion of Enterobacteriaceae can exacerbate disease (Garrett et al., 2010). However, the idea that dysbiosis is a biomarker of an underlying immunodeficiency suggests that potential treatment targets are not limited to the microbiota but could include other microbiome components, such as host factors that control the microbial environment (Litvak et al., 2018). Dysbiosis in the colon is often linked to a change in epithelial energy metabolism (Litvak et al., 2017), which raises the question of whether the underlying signaling pathways that control mitochondrial activity represent a target for dysbiosis therapy. During homeostasis, the PPAR-g signaling pathway maintains epithelial hypoxia (Byndloss et al., 2017), a key component of microbiotanourishing immunity in the colon. PPAR-g engages the transcription coactivator PGC-1a (peroxisome proliferator-activated receptor-gamma coactivator-1 alpha) to activate transcription of the SIRT3 gene, which encodes a protein deacetylase termed Sirtuin 3 (SIRT3) (Figure 2A) (Giralt et al., 2011). SIRT3 is localized to the mitochondria, where it deacetylates and activates a number of enzymes involved in oxidative phosphorylation (including the complex I subunit NDUFA9; Ahn et al., 2008), the tricarboxylic acid (TCA) cycle (including succinate dehydrogenase [SDHA]; Finley et al., 2011), and b-oxidation of fatty acids (including long-chain acyl-CoA dehydrogenase [LCAD]; Hirschey et al., 2010). The consequent increase in mitochondrial activity drives a high epithelial oxygen consumption, which maintains epithelial hypoxia (Zheng et al., 2015). In patients with ulcerative colitis, an inflammatory bowel disease of the colon, the PPAR-g signaling component of microbiota-nourishing immunity is impaired (Byndloss et al., 2019). Specifically, colonic epithelial cells from ulcerative colitis patients exhibit lower epithelial PPAR-g synthesis (Dubuquoy et al., 2003) and reduced mitochondrial activity (Roediger, 1980) compared to healthy individuals. The consequent changes in epithelial physiology are predicted to increase oxygenation of the epithelial surface, which would provide a mechanistic explanation for a disruption in anaerobiosis that is proposed to drive an expansion of Proteobacteria in the colon during ulcerative colitis (Rigottier-Gois, 2013). If these assumptions are correct, activation of epithelial PPAR-g signaling is projected to remediate dysbiosis in ulcerative colitis patients. Consistent with this hypothesis, a recent clinical study shows that treatment of ulcerative colitis patients with the PPAR-g agonist mesalazine (Rousseaux et al., 2013), which acts topically on the colonic epithelium (Zhou et al., 1999), is associated with lowering the abundance of Proteobacteria in the colonic microbiota (Xu et al., 2018)

(Figure 2A). Conversely, treatment of patients with drugs that inhibit the PPAR-g signaling pathway would be predicted to be associated with dysbiosis characterized by an expansion of Proteobacteria. Metformin, a first-line medication for the treatment of type 2 diabetes, reduces SIRT3 expression in the liver of patients (Buler et al., 2012) (Figure 2B). Microbiota profiling of feces from type 2 diabetes patients reveals that metformin treatment is associated with a significant increase in the abundance of Proteobacteria, including E. coli (Ejtahed et al., 2018; Wu et al., 2017), which is consistent with the idea that inhibiting the PPAR-g signaling pathway induces dysbiosis in the human colon. Collectively, the clinical observations discussed above support the idea that epithelial PPAR-g signaling is a promising therapeutic target for approaches to remediate dysbiosis in the colon, because it acts as a control switch that mediates the shift between homeostatic and dysbiotic microbial communities (Litvak et al., 2018). Targeting host components of microbiota-nourishing immunity might also be a viable strategy for treating dysbiosis at other mucosal surfaces, provided that we succeed in identifying the relevant host control mechanisms that are operational at those locations. Surprisingly, virulence factors of mucosal pathogens might provide the help we need to identify these treatment targets (Spiga and Winter, 2019). Pathogens as Tools to Study Microbiota-Nourishing Immunity Virulence factors enable pathogens to attach to or enter host cells, inhibit or evade the host’s immune responses, and obtain nutrients. Since colonization resistance is the first line of defense encountered by mucosal pathogens, it is predictable that some virulence factors enable pathogens to overcome niche preemption or niche modification mediated by our microbial organ (Figure 1B). Both pathogens and commensals employ antibacterial weaponry, such as colicins, microcins, or type VI secretion systems during their competition with other microbes for nutrients (Nedialkova et al., 2014; Sana et al., 2016; Sassone-Corsi et al., 2016). However, only pathogens use their virulence factors to elicit immune responses that generate symptoms of disease. An emerging idea in the field of bacterial pathogenesis suggests that the goal from the mucosal pathogen’s point of view is not to make us sick but to alter the host environment for the purpose of niche modification, thereby creating a habitat favorable for ecosystem invasion (Rohmer et al., 2011). Salmonella enterica serovar (S.) Typhimurium (family Enterobacteriaceae) is a wellstudied example of a how a facultative anaerobic enteric pathogen can use its virulence factors to overcome colonization resistance in the colon. Ecosystem invasion by S. Typhimurium is a two-stage process. The first stage is characterized by a decline in S. Typhimurium numbers, which is followed by a rapid pathogen expansion in the feces during the second stage. When the pathogen first enters the ecosystem, it faces the dilemma that the microbiota limits access to growth-limiting resources. Niche preemption against S. Typhimurium is mediated by endogenous Enterobacteriaceae, such as commensal E. coli, which compete with the pathogen for growth-limiting nutrients that support growth by aerobic respiration (Brugiroux et al., 2016; Litvak et al., 2019; Velazquez et al., 2019). In addition, the Clostridia-derived metabolite butyrate (Louis and Flint, Immunity 51, August 20, 2019 219

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Review Figure 3. S. Typhimurium Virulence Factors Facilitate Ecosystem Invasion by Eliciting Host Responses that Result in Niche Modification Ecosystem invasion by S. Typhimurium is a twostage process in which an initial decline of pathogen numbers (first stage) is followed by a pathogen expansion (second stage). The schematic on the top left depicts the mechanism through which Clostridia-mediated niche modification and Enterobacteriaceae-mediated niche preemption confer colonization resistance during the first phase of ecosystem invasion. The schematic on the top right depicts the chain of events through which virulence factors enable S. Typhimurium to overcome Clostridia-mediated niche modification during the second phase of ecosystem invasion. O2, oxygen; NO3 , nitrate; S4O62 , tetrathionate; CO2, carbon dioxide.

2009; Vital et al., 2014) promotes niche modification by preserving epithelial hypoxia (Donohoe et al., 2011), thereby limiting the pathogen’s access to epithelial-derived oxygen (Byndloss et al., 2017). Neither Enterobacteriaceae nor Clostridia alone are sufficient to confer niche protection, but germ-free mice associated with a mixture of both taxa acquire colonization resistance (Litvak et al., 2019). Thus, the pathogen is repelled by a combination of Enterobacteriaceae-mediated niche preemption and Clostridia-mediated niche modification (Figure 3). The consequent inability to access growth-limiting resources leads to an initial decline in pathogen numbers, which can lead to an extinction, an outcome that becomes more likely when the challenge dose is low (Velazquez et al., 2019). To overcome colonization resistance, a subset of the pathogen population uses its virulence factors to invade the intestinal mucosa (Tsolis et al., 1999), whereas the luminal pathogen population needs to persist until the ensuing host response paves the way for a S. Typhimurium expansion (Stecher et al., 2007), which marks the beginning of the second stage of ecosystem invasion. In genetically resistant hosts, bacteria in tissue are eventually cleared by sterilizing immunity, but in an attempt to protect the epithelial surface, neutrophils are recruited into the intestinal lumen, where they deplete Clostridia (Gill et al., 2012; Sekirov et al., 2010). In turn, a depletion of Clostridia from the microbiota lowers the concentration of butyrate, thereby impairing microbiota-nourishing immunity by increasing epithelial oxygenation in the colon, which enhances growth of the luminal S. Typhimurium population by aerobic respiration (Rivera-Cha´vez et al., 2016). The shift in metabolism that accompanies epithelial dysfunction results in a release of host-derived lactate (Gillis et al., 2018, 2019), a metabolite that neutralizes growth inhibition of S. Typhimurium by short-chain fatty acids (Bohnhoff et al., 1964). Reactive oxygen and nitrogen species produced by luminal phagocytes react to generate nitrate and tetrathionate, which further escalate S. Typhimurium growth 220 Immunity 51, August 20, 2019

by serving as electron acceptors for anaerobic respiration (Lopez et al., 2012, 2015; Winter et al., 2010). Aerobic and anaerobic respiration synergize to escalate an expansion of the luminal S. Typhimurium population (Figure 3), which is required for fecal-oral transmission of the pathogen (Rivera-Cha´vez et al., 2016). Thus, by mediating bacterial invasion of the intestinal mucosa, S. Typhimurium virulence factors trigger sterilizing immunity, which provides benefit by clearing the pathogen from tissue but comes at the cost of weakening microbiota-nourishing immunity. Through this complex chain of events, virulence factors enable S. Typhimurium to overcome colonization resistance by triggering host responses that culminate in niche modification of the luminal environment. Notably, S. Typhimurium virulence factors impair the host’s ability to limit the availability of respiratory electron acceptors, a host component of microbiota-nourishing immunity that supports the accessory digestive functions of the colonic microbiota. This example illustrates that pathogens target key components of microbiota-nourishing immunity, which makes them useful tools for identifying missing pieces in the microbiome puzzle (Spiga and Winter, 2019). Determining how virulence factors allow pathogens to overcome colonization resistance at body habitats outside the colon might help us identify accessory functions of the microbiota and the corresponding host mechanisms that maintain them. In turn, this information might aid in understanding what constitutes a healthy microbiome at the respective body habitat and illuminate how a specific taxa composition supports the benefit that the microbiota provides. Concluding Remarks The hope that by looking deep into the microbiota composition, technology-based empiricism will help us understand what constitutes a healthy microbiome has not yet come to fruition. However, a functional definition for homeostasis emerges when concepts from community ecology and immunology are incorporated into microbiome research. We propose that the microbiota and its host environment, two components of the microbiome, form a functional unit that confers colonization resistance

Immunity

Review against pathogens, thereby forming a separate branch of our immune system, termed microbiota-nourishing immunity. Dysbiosis is an immunodeficiency in which this non-specific immune function is weakened, whereas a healthy microbiome can be defined functionally as a state in which microbiota-nourishing immunity is intact. Here we raise the question whether the vision of microbiotanourishing immunity can pull microbiome research out of its pre-theory stage. The idea that host control over the microbial environment maintains core and accessory functions of the microbiota to preserve a healthy microbiome is supported by a body of data on bacterial communities from one body habitat, the colon. Great challenges remain in identifying accessory functions for other taxa (e.g., fungi, protozoa, or viruses) or for bacterial communities at other body habitats, but this also creates great opportunities for identifying host cell types and mechanisms involved in this branch of immunity, which could become therapeutic targets to strengthen microbiota-nourishing immunity at the respective body surfaces. We argue that supplementing technology-based empiricism with deductive logic, studies on the physiology of commensal microbes, and characterization of immune mechanisms and cell types that shape the microbial ecosystem will accelerate meeting these challenges and opportunities and help make the next decade a golden age of microbiome research. ACKNOWLEDGMENTS Y.L. was supported by Vaadia-BARD Postdoctoral Fellowship FI-505-2014. Work in A.J.B.’s lab is supported by USDA/NIFA award 2015-67015-22930 and by Public Health Service Grants AI044170, AI096528, AI112445, and AI112949.

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