Rhythmicity of intestinal IgA responses confers oscillatory commensal microbiota mutualism.

Interactions between the mammalian host and commensal microbiota are enforced through a range of immune responses that confer metabolic benefits and promote tissue health and homeostasis. Immunoglobulin A (IgA) responses directly determine the composition of commensal species that colonize the intestinal tract but require substantial metabolic resources to fuel antibody production by tissue-resident plasma cells. Here, we demonstrate that IgA responses are subject to diurnal regulation over the course of a circadian day. Specifically, the magnitude of IgA secretion, as well as the transcriptome of intestinal IgA+ plasma cells, was found to exhibit rhythmicity. Oscillatory IgA responses were found to be entrained by time of feeding and were also found to be in part coordinated by the plasma cell-intrinsic circadian clock via deletion of the master clock gene Arntl. Moreover, reciprocal interactions between the host and microbiota dictated oscillatory dynamics among the commensal microbial community and its associated transcriptional and metabolic activity in an IgA-dependent manner. Together, our findings suggest that circadian networks comprising intestinal IgA, diet, and the microbiota converge to align circadian biology in the intestinal tract and to ensure host-microbial mutualism.

Macrophage metabolism in the intestine is compartment specific and regulated by the microbiota.

Intestinal macrophages play a vital role in the maintenance of gut homeostasis through signals derived from the microbiota. We previously demonstrated that microbial-derived metabolites can shape the metabolic functions of macrophages. Here, we show that antibiotic-induced disruption of the intestinal microbiota dramatically alters both the local metabolite environment and the metabolic functions of macrophages in the colon. Broad-spectrum antibiotic administration in mice increased the expression of the large neutral amino acid transporter LAT1 and accordingly, amino acid uptake. Subsequently, antibiotic administration enhanced the metabolic functions of colonic macrophages, increasing phosphorylation of components of mammalian/mechanistic target of rapamycin signalling pathways, with increased expression of genes involved in glycolysis and oxidative phosphorylation (OXPHOS), increased mitochondrial function, increased rate of extracellular acidification (ECAR; measure of glycolysis) and increased rate of oxygen consumption (OCR; measure of OXPHOS). Small bowel macrophages were less metabolically active than their colonic counterparts, with macrophage metabolism in the small intestine being independent of the microbiota. Finally, we reveal tissue-resident Tim4+  CD4+ macrophages exhibit enhanced fatty acid uptake alongside reduced fatty acid synthesis compared to recruited macrophages. Thus, the microbiota shapes gut macrophage metabolism in a compartment-specific manner, with important implications for monocyte recruitment and macrophage differentiation.

The interplay between Trichuris and the microbiota.

Parasitic worms are amongst the most common pathogens to infect humans and have a long-established history of inflicting disease in their hosts. There is a large body of evidence that states intestine-dwelling helminths ensure their survival by influencing the host immune response against them. In recent years, it has become apparent that the large and diverse microbial communities that exist in the gastrointestinal (GI) tract of the host and within the parasite itself have a pivotal role in worm survival and persistence. Using a variety of mouse models (including laboratory, germ-free and rewilded mice), there have been new insights into how bacteria and worms interact with each other; this includes the discovery that Trichuris is unable to hatch and/or infect their host in the absence of bacteria, and that these worms contain a Trichuris-specific gut microbiota. These interactions are determined in part by the capacity of the host, gut microbiota and worms to communicate via metabolites such as butyrate, which are microbially derived and have known immunoregulatory properties. By exploring the contribution of gut bacteria to worm infections and the intricate relationship that exists between them, an exciting and emerging field in whipworm parasitology is established.

Microbiota Supplementation with Bifidobacterium and Lactobacillus Modifies the Preterm Infant Gut Microbiota and Metabolome: An Observational Study.

Supplementation with members of the early-life microbiota as "probiotics" is increasingly used in attempts to beneficially manipulate the preterm infant gut microbiota. We performed a large observational longitudinal study comprising two preterm groups: 101 infants orally supplemented with Bifidobacterium and Lactobacillus (Bif/Lacto) and 133 infants non-supplemented (control) matched by age, sex, and delivery method. 16S rRNA gene profiling on fecal samples (n = 592) showed a predominance of Bifidobacterium and a lower abundance of pathobionts in the Bif/Lacto group. Metabolomic analysis showed higher fecal acetate and lactate and a lower fecal pH in the Bif/Lacto group compared to the control group. Fecal acetate positively correlated with relative abundance of Bifidobacterium, consistent with the ability of the supplemented Bifidobacterium strain to metabolize human milk oligosaccharides into acetate. This study demonstrates that microbiota supplementation is associated with a Bifidobacterium-dominated preterm microbiota and gastrointestinal environment more closely resembling that of full-term infants.

Mucosal or systemic microbiota exposures shape the B cell repertoire.

Colonization by the microbiota causes a marked stimulation of B cells and induction of immunoglobulin, but mammals colonized with many taxa have highly complex and individualized immunoglobulin repertoires1,2. Here we use a simplified model of defined transient exposures to different microbial taxa in germ-free mice3 to deconstruct how the microbiota shapes the B cell pool and its functional responsiveness. We followed the development of the immunoglobulin repertoire in B cell populations, as well as single cells by deep sequencing. Microbial exposures at the intestinal mucosa generated oligoclonal responses that differed from those of germ-free mice, and from the diverse repertoire that was generated after intravenous systemic exposure to microbiota. The IgA repertoire-predominantly to cell-surface antigens-did not expand after dose escalation, whereas increased systemic exposure broadened the IgG repertoire to both microbial cytoplasmic and cell-surface antigens. These microbial exposures induced characteristic immunoglobulin heavy-chain repertoires in B cells, mainly at memory and plasma cell stages. Whereas sequential systemic exposure to different microbial taxa diversified the IgG repertoire and facilitated alternative specific responses, sequential mucosal exposure produced limited overlapping repertoires and the attrition of initial IgA binding specificities. This shows a contrast between a flexible response to systemic exposure with the need to avoid fatal sepsis, and a restricted response to mucosal exposure that reflects the generic nature of host-microbial mutualism in the mucosa.

Breast milk-derived human milk oligosaccharides promote Bifidobacterium interactions within a single ecosystem.

Diet-microbe interactions play an important role in modulating the early-life microbiota, with Bifidobacterium strains and species dominating the gut of breast-fed infants. Here, we sought to explore how infant diet drives distinct bifidobacterial community composition and dynamics within individual infant ecosystems. Genomic characterisation of 19 strains isolated from breast-fed infants revealed a diverse genomic architecture enriched in carbohydrate metabolism genes, which was distinct to each strain, but collectively formed a pangenome across infants. Presence of gene clusters implicated in digestion of human milk oligosaccharides (HMOs) varied between species, with growth studies indicating that within single infants there were differences in the ability to utilise 2'FL and LNnT HMOs between strains. Cross-feeding experiments were performed with HMO degraders and non-HMO users (using spent or 'conditioned' media and direct co-culture). Further 1H-NMR analysis identified fucose, galactose, acetate, and N-acetylglucosamine as key by-products of HMO metabolism; as demonstrated by modest growth of non-HMO users on spend media from HMO metabolism. These experiments indicate how HMO metabolism permits the sharing of resources to maximise nutrient consumption from the diet and highlights the cooperative nature of bifidobacterial strains and their role as 'foundation' species in the infant ecosystem. The intra- and inter-infant bifidobacterial community behaviour may contribute to the diversity and dominance of Bifidobacterium in early life and suggests avenues for future development of new diet and microbiota-based therapies to promote infant health.

Host-microbe interaction in the gastrointestinal tract.

The gastrointestinal tract is a highly complex organ in which multiple dynamic physiological processes are tightly coordinated while interacting with a dense and extremely diverse microbial population. From establishment in early life, through to host-microbe symbiosis in adulthood, the gut microbiota plays a vital role in our development and health. The effect of the microbiota on gut development and physiology is highlighted by anatomical and functional changes in germ-free mice, affecting the gut epithelium, immune system and enteric nervous system. Microbial colonisation promotes competent innate and acquired mucosal immune systems, epithelial renewal, barrier integrity, and mucosal vascularisation and innervation. Interacting or shared signalling pathways across different physiological systems of the gut could explain how all these changes are coordinated during postnatal colonisation, or after the introduction of microbiota into germ-free models. The application of cell-based in-vitro experimental systems and mathematical modelling can shed light on the molecular and signalling pathways which regulate the development and maintenance of homeostasis in the gut and beyond.

The liver may act as a firewall mediating mutualism between the host and its gut commensal microbiota.

A prerequisite for establishment of mutualism between the host and the microbial community that inhabits the large intestine is the stringent mucosal compartmentalization of microorganisms. Microbe-loaded dendritic cells trafficking through lymphatics are arrested at the mesenteric lymph nodes, which constitute the firewall of the intestinal lymphatic circulation. We show in different mouse models that the liver, which receives the intestinal venous blood circulation, forms a vascular firewall that captures gut commensal bacteria entering the bloodstream during intestinal pathology. Phagocytic Kupffer cells in the liver of mice clear commensals from the systemic vasculature independently of the spleen through the liver's own arterial supply. Damage to the liver firewall in mice impairs functional clearance of commensals from blood, despite heightened innate immunity, resulting in spontaneous priming of nonmucosal immune responses through increased systemic exposure to gut commensals. Systemic immune responses consistent with increased extraintestinal commensal exposure were found in humans with liver disease (nonalcoholic steatohepatitis). The liver may act as a functional vascular firewall that clears commensals that have penetrated either intestinal or systemic vascular circuits.

Mice overexpressing BAFF develop a commensal flora-dependent, IgA-associated nephropathy.

B cell activation factor of the TNF family (BAFF) is a potent B cell survival factor. BAFF overexpressing transgenic mice (BAFF-Tg mice) exhibit features of autoimmune disease, including B cell hyperplasia and hypergammaglobulinemia, and develop fatal nephritis with age. However, basal serum IgA levels are also elevated, suggesting that the pathology in these mice may be more complex than initially appreciated. Consistent with this, we demonstrate here that BAFF-Tg mice have mesangial deposits of IgA along with high circulating levels of polymeric IgA that is aberrantly glycosylated. Renal disease in BAFF-Tg mice was associated with IgA, because serum IgA was highly elevated in nephritic mice and BAFF-Tg mice with genetic deletion of IgA exhibited less renal pathology. The presence of commensal flora was essential for the elevated serum IgA phenotype, and, unexpectedly, commensal bacteria-reactive IgA antibodies were found in the blood. These data illustrate how excess B cell survival signaling perturbs the normal balance with the microbiota, leading to a breach in the normal mucosal-peripheral compartmentalization. Such breaches may predispose the nonmucosal system to certain immune diseases. Indeed, we found that a subset of patients with IgA nephropathy had elevated serum levels of a proliferation inducing ligand (APRIL), a cytokine related to BAFF. These parallels between BAFF-Tg mice and human IgA nephropathy may provide a new framework to explore connections between mucosal environments and renal pathology.

Intestinal bacterial colonization induces mutualistic regulatory T cell responses.

Mammals harbor a dense commensal microbiota in the colon. Regulatory T (Treg) cells are known to limit microbe-triggered intestinal inflammation and the CD4+ T cell compartment is shaped by the presence of particular microbes or bacterial compounds. It is, however, difficult to distinguish whether these effects reflect true mutualistic immune adaptation to intestinal colonization or rather idiosyncratic immune responses. To investigate truly mutualistic CD4+ T cell adaptation, we used the altered Schaedler flora (ASF). Intestinal colonization resulted in activation and de novo generation of colonic Treg cells. Failure to activate Treg cells resulted in the induction of T helper 17 (Th17) and Th1 cell responses, which was reversed by wild-type Treg cells. Efficient Treg cell induction was also required to maintain intestinal homeostasis upon dextran sulfate sodium-mediated damage in the colon. Thus, microbiota colonization-induced Treg cell responses are a fundamental intrinsic mechanism to induce and maintain host-intestinal microbial T cell mutualism.

Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses.

The lower intestine of adult mammals is densely colonized with nonpathogenic (commensal) microbes. Gut bacteria induce protective immune responses, which ensure host-microbial mutualism. The continuous presence of commensal intestinal bacteria has made it difficult to study mucosal immune dynamics. Here, we report a reversible germ-free colonization system in mice that is independent of diet or antibiotic manipulation. A slow (more than 14 days) onset of a long-lived (half-life over 16 weeks), highly specific anticommensal immunoglobulin A (IgA) response in germ-free mice was observed. Ongoing commensal exposure in colonized mice rapidly abrogated this response. Sequential doses lacked a classical prime-boost effect seen in systemic vaccination, but specific IgA induction occurred as a stepwise response to current bacterial exposure, such that the antibody repertoire matched the existing commensal content.

Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism.

Commensal bacteria in the lower intestine of mammals are 10 times as numerous as the body's cells. We investigated the relative importance of different immune mechanisms in limiting the spread of the intestinal microbiota. Here, we reveal a flexible continuum between innate and adaptive immune function in containing commensal microbes. Mice deficient in critical innate immune functions such as Toll-like receptor signaling or oxidative burst production spontaneously produce high-titer serum antibodies against their commensal microbiota. These antibody responses are functionally essential to maintain host-commensal mutualism in vivo in the face of innate immune deficiency. Spontaneous hyper-activation of adaptive immunity against the intestinal microbiota, secondary to innate immune deficiency, may clarify the underlying mechanisms of inflammatory diseases where immune dysfunction is implicated.