Emerging evidence suggests that the effect of dietary intake on human health and disease is linked to both the immune system and the microbiota. Yet, we lack an integrated mechanistic model for how these three complex systems relate, limiting our ability to understand and treat chronic and infectious disease. Here, we review recent findings at the interface of microbiology, immunology, and nutrition, with an emphasis on experimentally tractable models and hypothesis-driven mechanistic work. We outline emerging mechanistic concepts and generalizable approaches to bridge the gap between microbial ecology and molecular mechanism. These set the stage for a new era of precision human nutrition informed by a deep and comprehensive knowledge of the diverse cell types in and on the human body.

Despite the recognition, nearly a century ago, that the human microbiome plays a clinically relevant role in drug disposition, mechanistic insights, and translational applications are still limited. Here, we highlight the recent re-emergence of "pharmacomicrobiomics," which seeks to understand how inter-individual variations in the microbiome shape drug efficacy and side effect profiles. Multiple bacterial species, genes, and enzymes have already been implicated in the direct biotransformation of drugs, both from targeted case studies and from systematic computational and experimental analyses. Indirect mechanisms are also at play; for example, microbial interactions with the host immune system can have broad effects on immunomodulatory drugs. Finally, we discuss multiple emerging strategies for the precise manipulation of complex microbial communities to improve treatment outcomes. In the coming years, we anticipate a shift toward a more comprehensive view of precision medicine that encompasses our human and microbial genomes and their combined metabolic activities.

Eggerthella lenta is a prevalent human gut Actinobacterium implicated in drug, dietary phytochemical, and bile acid metabolism and associated with multiple human diseases. No genetic tools are currently available for the direct manipulation of E. lenta. Here, we construct shuttle vectors and develop methods to transform E. lenta and other Coriobacteriia. With these tools, we characterize endogenous E. lenta constitutive and inducible promoters using a reporter system and construct inducible expression systems, enabling tunable gene regulation. We also achieve genome editing by harnessing an endogenous type I-C CRISPR-Cas system. Using these tools to perform genetic knockout and complementation, we dissect the functions of regulatory proteins and enzymes involved in catechol metabolism, revealing a previously unappreciated family of membrane-spanning LuxR-type transcriptional regulators. Finally, we employ our genetic toolbox to study the effects of E. lenta genes on mammalian host biology. By greatly expanding our ability to study and engineer gut Coriobacteriia, these tools will reveal mechanistic details of host-microbe interactions and provide a roadmap for genetic manipulation of other understudied human gut bacteria.

Bacterial activation of T helper 17 (Th17) cells exacerbates mouse models of autoimmunity, but how human-associated bacteria impact Th17-driven disease remains elusive. We show that human gut Actinobacterium Eggerthella lenta induces intestinal Th17 activation by lifting inhibition of the Th17 transcription factor Rorγt through cell- and antigen-independent mechanisms. E. lenta is enriched in inflammatory bowel disease (IBD) patients and worsens colitis in a Rorc-dependent manner in mice. Th17 activation varies across E. lenta strains, which is attributable to the cardiac glycoside reductase 2 (Cgr2) enzyme. Cgr2 is sufficient to induce interleukin (IL)-17a, a major Th17 cytokine. cgr2+ E. lenta deplete putative steroidal glycosides in pure culture; related compounds are negatively associated with human IBD severity. Finally, leveraging the sensitivity of Cgr2 to dietary arginine, we prevented E. lenta-induced intestinal inflammation in mice. Together, these results support a role for human gut bacterial metabolism in driving Th17-dependent autoimmunity.

Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allow E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest.

Immunomodulatory drugs can inhibit bacterial growth, yet their mechanism of action, spectrum, and clinical relevance remain unknown. Methotrexate (MTX), a first-line rheumatoid arthritis (RA) treatment, inhibits mammalian dihydrofolate reductase (DHFR), but whether it directly impacts gut bacteria is unclear. We show that MTX broadly alters the human gut microbiota. Drug sensitivity varied across strains, but the mechanism of action against DHFR appears conserved between mammalian and bacterial cells. RA patient microbiotas were sensitive to MTX, and changes in gut bacterial taxa and gene family abundance were distinct between responders and non-responders. Transplantation of post-treatment samples into germ-free mice given an inflammatory trigger led to reduced immune activation relative to pre-treatment controls, enabling identification of MTX-modulated bacterial taxa associated with intestinal and splenic immune cells. Thus, conservation in cellular pathways across domains of life can result in broad off-target drug effects on the human gut microbiota with consequences for immune function.

Very low-carbohydrate, high-fat ketogenic diets (KDs) induce a pronounced shift in metabolic fuel utilization that elevates circulating ketone bodies; however, the consequences of these compounds for host-microbiome interactions remain unknown. Here, we show that KDs alter the human and mouse gut microbiota in a manner distinct from high-fat diets (HFDs). Metagenomic and metabolomic analyses of stool samples from an 8-week inpatient study revealed marked shifts in gut microbial community structure and function during the KD. Gradient diet experiments in mice confirmed the unique impact of KDs relative to HFDs with a reproducible depletion of bifidobacteria. In vitro and in vivo experiments showed that ketone bodies selectively inhibited bifidobacterial growth. Finally, mono-colonizations and human microbiome transplantations into germ-free mice revealed that the KD-associated gut microbiota reduces the levels of intestinal pro-inflammatory Th17 cells. Together, these results highlight the importance of trans-kingdom chemical dialogs for mediating the host response to dietary interventions.

Chronic inflammation is a contributing factor to most life-shortening human diseases. However, the molecular and cellular mechanisms that sustain chronic inflammatory responses remain poorly understood, making it difficult to treat this deleterious condition. Using a mouse model of age-dependent inflammation that results from a deficiency in miR-146a, we demonstrate that miR-155 contributed to the progressive inflammatory disease that emerged as Mir146a(-/-) mice grew older. Upon analyzing lymphocytes from inflamed versus healthy middle-aged mice, we found elevated numbers of T follicular helper (Tfh) cells, germinal center (GC) B cells, and autoantibodies, all occurring in a miR-155-dependent manner. Further, Cd4-cre Mir155(fl/fl) mice were generated and demonstrated that miR-155 functions in T cells, in addition to its established role in B cells, to promote humoral immunity in a variety of contexts. Taken together, our study discovers that miR-146a and miR-155 counterregulate Tfh cell development that drives aberrant GC reactions during chronic inflammation.

Human gut bacteria perform diverse metabolic functions with consequences for host health. The prevalent and disease-linked Actinobacterium Eggerthella lenta performs several unusual chemical transformations, but it does not metabolize sugars and its core growth strategy remains unclear. To obtain a comprehensive view of the metabolic network of E. lenta, we generated several complementary resources: defined culture media, metabolomics profiles of strain isolates, and a curated genome-scale metabolic reconstruction. Stable isotope-resolved metabolomics revealed that E. lenta uses acetate as a key carbon source while catabolizing arginine to generate ATP, traits which could be recapitulated in silico by our updated metabolic model. We compared these in vitro findings with metabolite shifts observed in E. lenta-colonized gnotobiotic mice, identifying shared signatures across environments and highlighting catabolism of the host signaling metabolite agmatine as an alternative energy pathway. Together, our results elucidate a distinctive metabolic niche filled by E. lenta in the gut ecosystem. Our culture media formulations, atlas of metabolomics data, and genome-scale metabolic reconstructions form a freely available collection of resources to support further study of the biology of this prevalent gut bacterium.

FLT3-ITD⁺ acute myeloid leukemia (AML) accounts for ∼25% of all AML cases and is a subtype that carries a poor prognosis. microRNA-155 (miR-155) is specifically overexpressed in FLT3-ITD⁺ AML compared with FLT3 wild-type (FLT3-WT) AML and is critical for the growth of FLT3-ITD⁺ AML cells in vitro. However, miR-155's role in regulating FLT3-ITD-mediated disease in vivo remains unclear. In this study, we used a genetic mouse model to determine whether miR-155 influences the development of FLT3-ITD-induced myeloproliferative disease. Results indicate that miR-155 promotes FLT3-ITD-induced myeloid expansion in the bone marrow, spleen, and peripheral blood. Mechanistically, miR-155 increases proliferation of the hematopoietic stem and progenitor cell compartments by reducing the growth-inhibitory effects of the interferon (IFN) response, and this involves targeting of Cebpb. Consistent with our observations in mice, primary FLT3-ITD⁺ AML clinical samples have significantly higher miR-155 levels and a lower IFN response compared with FLT3-WT AML samples. Further, inhibition of miR-155 in FLT3-ITD⁺ AML cell lines using CRISPR/Cas9, or primary FLT3-ITD⁺ AML samples using locked nucleic acid antisense inhibitors, results in an elevated IFN response and reduces colony formation. Altogether, our data reveal that miR-155 collaborates with FLT3-ITD to promote myeloid cell expansion in vivo and that this involves a multitarget mechanism that includes repression of IFN signaling.