An immune-competent human gut microphysiological system enables inflammation-modulation by Faecalibacterium prausnitzii.

Crosstalk of microbes with human gut epithelia and immune cells is crucial for gut health. However, there is no existing system for a long-term co-culture of human innate immune cells with epithelium and oxygen-intolerant commensal microbes, hindering the understanding of microbe-immune interactions in a controlled manner. Here, we established a gut epithelium-microbe-immune (GuMI) microphysiological system to maintain the long-term continuous co-culture of Faecalibacterium prausnitzii/Faecalibacterium duncaniae with colonic epithelium, antigen-presenting cells (APCs, herein dendritic cells and macrophages), and CD4+ naive T cells circulating underneath the colonic epithelium. In GuMI-APC condition, multiplex cytokine assays suggested that APCs contribute to the elevated level of cytokines and chemokines secreted into both apical and basolateral compartments compared to GuMI condition that lacks APC. In GuMI-APC with F. prausnitzii (GuMI-APC-FP), F. prausnitzii increased the transcription of pro-inflammatory genes such as toll-like receptor 1 (TLR1) and interferon alpha 1 (IFNA1) in the colonic epithelium, without a significant effect on cytokine secretion, compared to the GuMI-APC without bacteria (GuMI-APC-NB). In contrast, in the presence of CD4+ naive T cells (GuMI-APCT-FP), TLR1, IFNA1, and IDO1 transcription levels decreased with a simultaneous increase in F. prausnitzii-induced secretion of pro-inflammatory cytokines (e.g., IL8) compared to GuMI-APC-FP that lacks T cells. These results highlight the contribution of individual innate immune cells in regulating the immune response triggered by the gut commensal F. prausnitzii. The integration of defined populations of immune cells in the gut microphysiological system demonstrated the usefulness of GuMI physiomimetic platform to study microbe-epithelial-immune interactions in healthy and disease conditions.

An immune-competent human gut microphysiological system enables inflammation-modulation of Faecalibacterium prausnitzii.

Crosstalk of microbes with human gut epithelia and immune cells is crucial for gut health. However, there is no existing system for a long-term co-culture of human innate immune cells with epithelium and oxygen-intolerant commensal microbes, hindering the understanding of microbe-immune interactions in a controlled manner. Here, we establish a gut epithelium-microbe-immune microphysiological system to maintain the long-term continuous co-culture of Faecalibacterium prausnitzii/Faecalibacterium duncaniae with colonic epithelium, antigen-presenting cells (APCs, herein dendritic cells and macrophages), with CD4+ naïve T cells circulating underneath the colonic epithelium. Multiplex cytokine assays suggested that APCs contribute to the elevated level of cytokines and chemokines being secreted into both apical and basolateral compartments. In contrast, the absence of APCs does not allow reliable detection of these cytokines. In the presence of APCs, F. prausnitzii increased the transcription of pro-inflammatory genes such as toll-like receptor 1 (TLR1) and interferon alpha 1 (IFNA1) in the colonic epithelium, but no significant change on the secreted cytokines. In contrast, integration of CD4+ naïve T cells reverses this effect by decreasing the transcription of TLR1, IFNA1, and indoleamine 2,3-dioxygenase, and increasing the F. prausnitzii-induced secretion of pro-inflammatory cytokines such as IL-8, MCP-1/CCL2, and IL1A. These results highlight the contribution of individual innate immune cells in the regulation of the immune response triggered by the gut commensal F. prausnitzii. The successful integration of defined populations of immune cells in this gut microphysiological system demonstrated the usefulness of the GuMI physiomimetic platform to study microbe-epithelial-immune interactions in health and disease.

Primary human colonic mucosal barrier crosstalk with super oxygen-sensitive Faecalibacterium prausnitzii in continuous culture.

BACKGROUND: The gut microbiome plays an important role in human health and disease. Gnotobiotic animal and in vitro cell-based models provide some informative insights into mechanistic crosstalk. However, there is no existing system for a long-term co-culture of a human colonic mucosal barrier with super oxygen-sensitive commensal microbes, hindering the study of human-microbe interactions in a controlled manner. METHODS: Here, we investigated the effects of an abundant super oxygen-sensitive commensal anaerobe, Faecalibacterium prausnitzii, on a primary human mucosal barrier using a Gut-MIcrobiome (GuMI) physiome platform that we designed and fabricated. FINDINGS: Long-term continuous co-culture of F. prausnitzii for two days with colon epithelia, enabled by continuous flow of completely anoxic apical media and aerobic basal media, resulted in a strictly anaerobic apical environment fostering growth of and butyrate production by F. prausnitzii, while maintaining a stable colon epithelial barrier. We identified elevated differentiation and hypoxia-responsive genes and pathways in the platform compared with conventional aerobic static culture of the colon epithelia, attributable to a combination of anaerobic environment and continuous medium replenishment. Furthermore, we demonstrated anti-inflammatory effects of F. prausnitzii through HDAC and the TLR-NFKB axis. Finally, we identified that butyrate largely contributes to the anti-inflammatory effects by downregulating TLR3 and TLR4. CONCLUSIONS: Our results are consistent with some clinical observations regarding F. prausnitzii, thus motivating further studies employing this platform with more complex engineered colon tissues for understanding the interaction between the human colonic mucosal barrier and microbiota, pathogens, or engineered bacteria.

Author Correction: Genetic circuit design automation for the gut resident species Bacteroides thetaiotaomicron.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic circuit design automation for the gut resident species Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron is a human-associated bacterium that holds promise for delivery of therapies in the gut microbiome1. Therapeutic bacteria would benefit from the ability to turn on different programs of gene expression in response to conditions inside and outside of the gut; however, the availability of regulatory parts, and methods to combine them, have been limited in B. thetaiotaomicron2-5. We report implementation of Cello circuit design automation software6 for this species. First, we characterize a set of genome-integrated NOT/NOR gates based on single guide RNAs (CRISPR-dCas9) to inform a Bt user constraint file (UCF) for Cello. Then, logic circuits are designed to integrate sensors that respond to bile acid and anhydrotetracycline (aTc), including one created to distinguish between environments associated with bioproduction, the human gut, and after release. This circuit was found to be stable under laboratory conditions for at least 12 days and to function in bacteria associated with a primary colonic epithelial monolayer in an in vitro human gut model system.

Gut-Liver Physiomimetics Reveal Paradoxical Modulation of IBD-Related Inflammation by Short-Chain Fatty Acids.

Although the association between the microbiome and IBD and liver diseases is known, the cause and effect remain elusive. By connecting human microphysiological systems of the gut, liver, and circulating Treg and Th17 cells, we created a multi-organ model of ulcerative colitis (UC) ex vivo. The approach shows microbiome-derived short-chain fatty acids (SCFAs) to either improve or worsen UC severity, depending on the involvement of effector CD4 T cells. Using multiomics, we found SCFAs increased production of ketone bodies, glycolysis, and lipogenesis, while markedly reducing innate immune activation of the UC gut. However, during acute T cell-mediated inflammation, SCFAs exacerbated CD4+ T cell-effector function, partially through metabolic reprograming, leading to gut barrier disruption and hepatic injury. These paradoxical findings underscore the emerging utility of human physiomimetic technology in combination with systems immunology to study causality and the fundamental entanglement of immunity, metabolism, and tissue homeostasis.

Electrophoresis of cell membrane heparan sulfate regulates galvanotaxis in glial cells.

Endogenous electric fields modulate many physiological processes by promoting directional migration, a process known as galvanotaxis. Despite the importance of galvanotaxis in development and disease, the mechanism by which cells sense and migrate directionally in an electric field remains unknown. Here, we show that electrophoresis of cell surface heparan sulfate (HS) critically regulates this process. HS was found to be localized at the anode-facing side in fetal neural progenitor cells (fNPCs), fNPC-derived astrocytes and brain tumor-initiating cells (BTICs), regardless of their direction of galvanotaxis. Enzymatic removal of HS and other sulfated glycosaminoglycans significantly abolished or reversed the cathodic response seen in fNPCs and BTICs. Furthermore, Slit2, a chemorepulsive ligand, was identified to be colocalized with HS in forming a ligand gradient across cellular membranes. Using both imaging and genetic modification, we propose a novel mechanism for galvanotaxis in which electrophoretic localization of HS establishes cell polarity by functioning as a co-receptor and provides repulsive guidance through Slit-Robo signaling.

Cellular microenvironment modulates the galvanotaxis of brain tumor initiating cells.

Galvanotaxis is a complex process that represents the collective outcome of various contributing mechanisms, including asymmetric ion influxes, preferential activation of voltage-gated channels, and electrophoretic redistribution of membrane components. While a large number of studies have focused on various up- and downstream signaling pathways, little is known about how the surrounding microenvironment may interact and contribute to the directional response. Using a customized galvanotaxis chip capable of carrying out experiments in both two- and three-dimensional microenvironments, we show that cell-extracellular matrix (ECM) interactions modulate the galvanotaxis of brain tumor initiating cells (BTICs). Five different BTICs across three different glioblastoma subtypes were examined and shown to all migrate toward the anode in the presence of a direct-current electric field (dcEF) when cultured on a poly-L-ornithine/laminin coated surface, while the fetal-derived neural progenitor cells (fNPCs) migrated toward the cathode. Interestingly, when embedded in a 3D ECM composed of hyaluronic acid and collagen, BTICs exhibited opposite directional response and migrated toward the cathode. Pharmacological inhibition against a panel of key molecules involved in galvanotaxis further revealed the mechanistic differences between 2- and 3D galvanotaxis in BTICs. Both myosin II and phosphoinositide 3-kinase (PI3K) were found to hold strikingly different roles in different microenvironments.

The influence of electric field and confinement on cell motility.

The ability of cells to sense and respond to endogenous electric fields is important in processes such as wound healing, development, and nerve regeneration. In cell culture, many epithelial and endothelial cell types respond to an electric field of magnitude similar to endogenous electric fields by moving preferentially either parallel or antiparallel to the field vector, a process known as galvanotaxis. Here we report on the influence of dc electric field and confinement on the motility of fibroblast cells using a chip-based platform. From analysis of cell paths we show that the influence of electric field on motility is much more complex than simply imposing a directional bias towards the cathode or anode. The cell velocity, directedness, as well as the parallel and perpendicular components of the segments along the cell path are dependent on the magnitude of the electric field. Forces in the directions perpendicular and parallel to the electric field are in competition with one another in a voltage-dependent manner, which ultimately govern the trajectories of the cells in the presence of an electric field. To further investigate the effects of cell reorientation in the presence of a field, cells are confined within microchannels to physically prohibit the alignment seen in 2D environment. Interestingly, we found that confinement results in an increase in cell velocity both in the absence and presence of an electric field compared to migration in 2D.

Amphiphilic peptide-polymer conjugates based on the coiled-coil helix bundle.

Amphiphilic peptide-polymer conjugates can lead to hierarchically structured, biomolecular materials. Because the peptide structure determines the size, shape, and intermolecular interactions of these building blocks, systematic understanding of how the peptide structure and functionality are affected upon implementing hydrophobicity is required to direct their assemblies in solution and in the solid state. However, depending on the peptide sequence and native structure, previous studies have shown that the hydrophobic moieties affect peptide structures differently. Here, we present a solution study of amphiphilic peptide-polymer conjugates, where a hydrophobic polymer, polystyrene, is covalently linked to the N-terminus of a coiled-coil helix bundle-forming peptide. The effect of conjugated hydrophobic polymers on the peptide secondary and tertiary structures was examined using two types of model, coiled-coil helix bundles. In particular, the integrity of the binding pocket within the helix bundle upon hydrophobic polymer conjugation was evaluated. Upon attachment of polystyrene to the peptide N-terminus, the coiled-coil helices partially unfolded and functionality within the bundle core was inhibited. These observations are attributed to favorable interactions between hydrophobic residues with the PS block at the peptide-polymer interface that lead to rearrangement of peptide residues and consequently, unfolding of peptide structures. Thus, the hydrophobicity of the covalently linked polymers modifies the conjugates' architecture, size, and shape and may be used to tailor the assembly and disassembly process. Furthermore, the hydrophobicity of the covalently linked polymer needs to be taken into consideration to maintain the built-in functionalities of protein motifs when constructing amphiphilic peptide-polymer conjugates.