Guidelines and definitions for research on epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.

RET overactivation leads to concurrent Hirschsprung disease and intestinal ganglioneuromas.

Appropriately balanced RET signaling is of crucial importance during embryonic neural crest cell migration, proliferation and differentiation. RET deficiency, for example, leads to intestinal aganglionosis (Hirschsprung disease), whereas overactive RET can lead to multiple endocrine neoplasia (MEN) syndromes. Some RET mutations are associated with both intestinal aganglionosis and MEN-associated tumors. This seemingly paradoxical occurrence has led to speculation of a 'Janus mutation' in RET that causes overactivation or impairment of RET activity depending on the cellular context. Using an intestinal catenary culture system to test the effects of GDNF-mediated RET activation, we demonstrate the concurrent development of distal colonic aganglionosis and intestinal ganglioneuromas. Interestingly, the tumors induced by GDNF stimulation contain enteric neuronal progenitors capable of reconstituting an enteric nervous system when transplanted into a normal developmental environment. These results suggest that a Janus mutation may not be required to explain co-existing Hirschsprung disease and MEN-associated tumors, but rather that RET overstimulation alone is enough to cause both phenotypes. The results also suggest that reprogramming tumor cells toward non-pathological fates may represent a possible therapeutic avenue for MEN-associated neoplasms.

The Somatic Mutation Paradigm in Congenital Malformations: Hirschsprung Disease as a Model.

Patients with Hirschsprung disease (HSCR) do not always receive a genetic diagnosis after routine screening in clinical practice. One of the reasons for this could be that the causal mutation is not present in the cell types that are usually tested-whole blood, dermal fibroblasts or saliva-but is only in the affected tissue. Such mutations are called somatic, and can occur in a given cell at any stage of development after conception. They will then be present in all subsequent daughter cells. Here, we investigated the presence of somatic mutations in HSCR patients. For this, whole-exome sequencing and copy number analysis were performed in DNA isolated from purified enteric neural crest cells (ENCCs) and blood or fibroblasts of the same patient. Variants identified were subsequently validated by Sanger sequencing. Several somatic variants were identified in all patients, but causative mutations for HSCR were not specifically identified in the ENCCs of these patients. Larger copy number variants were also not found to be specific to ENCCs. Therefore, we believe that somatic mutations are unlikely to be identified, if causative for HSCR. Here, we postulate various modes of development following the occurrence of a somatic mutation, to describe the challenges in detecting such mutations, and hypothesize how somatic mutations may contribute to 'missing heritability' in developmental defects.

Fine scale differences within the vagal neural crest for enteric nervous system formation.

The enteric nervous system is mostly derived from vagal neural crest (NC) cells adjacent to somites (s)1-7. We used in ovo focal fluorescent vital dyes and focal electroporation of fluorophore-encoding plasmids in quail embryos to investigate NC cell migration to the foregut initially and later throughout the entire gut. NC cells of different somite-level origins were largely separate until reaching the foregut at about QE2.5, when all routes converged. By QE3.5, NC cells of different somite-levels became mixed, although s1-s2 NC cells were mainly confined to rostral foregut. Mid-vagal NC-derived cells (s3 and s4 level) arrived earliest at the foregut, and occurred in greatest number. By QE6.5 ENS was present from foregut to hindgut. Mid-vagal NC-derived cells occurred in greatest numbers from foregut to distal hindgut. NC-derived cells of s2, s5, and s6 levels were fewer and were widely distributed but were never observed in the distal hindgut. Rostro-vagal (s1) and caudo-vagal (s7) levels were few and restricted to the foregut. Single somite levels of quail neural tube/NC from s1 to s8 were combined with chick aneural ChE4.5 midgut and hindgut and the ensemble was grown on the chorio-allantoic membrane for 6 days. This tests ENS-forming competence in the absence of intra-segmental competition between NC cells, of differential influences of segmental paraxial tissues, and of positional advantage. All vagal NC-levels, but not s8 level, furnished enteric plexuses in the recipient gut, but the density of both ENS cells in total and neurons was highest from mid-vagal level donors, as was the length colonised. We conclude that the fate and competence for ENS formation of vagal NC sub-levels is not uniform over the vagal level but is biased to favour mid-vagal levels. Overviewing this and prior studies suggests the vagal region is, as in its traditional sense, a natural unit but with complex sub-divisions.

The enteric neural crest progressively loses capacity to form enteric nervous system.

Cells of the vagal neural crest (NC) form most of the enteric nervous system (ENS) by a colonising wave in the embryonic gut, with high cell proliferation and differentiation. Enteric neuropathies have an ENS deficit and cell replacement has been suggested as therapy. This would be performed post-natally, which raises the question of whether the ENS cell population retains its initial ENS-forming potential with age. We tested this on the avian model in organ culture in vitro (3 days) using recipient aneural chick midgut/hindgut combined with ENS-donor quail midgut or hindgut of ages QE5 to QE10. ENS cells from young donor tissues (≤ QE6) avidly colonised the aneural recipient, but this capacity dropped rapidly 2-3 days after the transit of the ENS cell wavefront. This loss in capability was autonomous to the ENS population since a similar decline was observed in ENS cells isolated by HNK1 FACS. Using QE5, 6, 8 and 10 midgut donors and extending the time of assay to 8 days in chorio-allantoic membrane grafts did not produce 'catch up' colonisation. NC-derived cells were counted in dissociated quail embryo gut and in transverse sections of chick embryo gut using NC, neuron and glial marker antibodies. This showed that the decline in ENS-forming ability correlated with a decrease in proportion of ENS cells lacking both neuronal and glial differentiation markers, but there were still large numbers of such cells even at stages with low colonisation ability. Moreover, ENS cells in small numbers from young donors were far superior in colonisation ability to larger numbers of apparently undifferentiated cells from older donors. This suggests that the decline of ENS-forming ability has both quantitative and qualitative aspects. In this case, ENS cells for cell therapies should aim to replicate the embryonic ENS stage rather than using post-natal ENS stem/progenitor cells.

Stochastic clonal expansion of "superstars" enhances the reserve capacity of enteric nervous system precursor cells.

We quantified cell population increase in the quail embryo enteric nervous system (ENS) from E2.5 (about 1500 cells) to E12 (about 8 million cells). We then probed ENS proliferative capacity by grafting to the chorio-allantoic membrane large (600 cells) and small (40 cells) populations of enteric neural crest (ENC) cells with aneural gut. This demonstrated that ENC cells show an extremely high capacity to regulate their proliferation while forming the ENS. Previous mathematical models and clonal label experiments revealed that a few dominant ENS "superstar" cell clones emerge but most clones are small. The model implied that "superstars" arise stochastically, but the same outcome could arise if "superstars" were pre-determined. We investigated these two modes mathematically and by grafting experiments with large and small numbers of ENCs, each including one EGFP-labelled ENC cell. The stochastic model predicts that the frequency of "superstar" detection increases as the ENC population decreases, the pre-determined model does not. Experimentally, as predicted by the stochastic model, the frequency of "superstar" detection increased with small ENC cell number. We conclude that ENS "superstar" clones achieve this status stochastically. Clonal dominance implies that clonal diversity is greatly reduced and in this case, somatic mutations may affect the phenotype. We suggest that somatic mutations coupled with loss of clonal diversity may contribute to variable penetrance and expressivity in individuals with genetically identical ENS pathologies.

Deficiency of the Wnt receptor Ryk causes multiple cardiac and outflow tract defects.

Ryk is a member of the receptor tyrosine kinase (RTK) family of proteins that control and regulate cellular processes. It is distinguished by binding Wnt ligands and having no detectable intrinsic protein tyrosine kinase activity suggesting Ryk is a pseudokinase. Here, we show an essential role for Ryk in directing morphogenetic events required for normal cardiac development through the examination of Ryk-deficient mice. We employed vascular corrosion casting, vascular perfusion with contrast dye, and immunohistochemistry to characterize cardiovascular and pharyngeal defects in Ryk-/- embryos. Ryk-/- mice exhibit a variety of malformations of the heart and outflow tract that resemble human congenital heart defects. This included stenosis and interruption of the aortic arch, ventriculoarterial malalignment, ventricular septal defects and abnormal pharyngeal arch artery remodelling. This study therefore defines a key intersection between a subset of growth factor receptors involved in planar cell polarity signalling, the Wnt family and mammalian cardiovascular development.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies.

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

Differential Clonal Expansion in an Invading Cell Population: Clonal Advantage or Dumb Luck?

In neoplastic cell growth, clones and subclones are variable both in size and mutational spectrum. The largest of these clones are believed to represent those cells with mutations that make them the most "fit," in a Darwinian sense, for expansion in their microenvironment. Thus, the degree of quantitative clonal expansion is regarded as being determined by innate qualitative differences between the cells that originate each clone. Here, using a combination of mathematical modelling and clonal labelling experiments applied to the developmental model system of the forming enteric nervous system, we describe how cells which are qualitatively identical may consistently produce clones of dramatically different sizes: most clones are very small while a few clones we term "superstars" contribute most of the cells to the final population. The basis of this is minor stochastic variations ("luck") in the timing and direction of movement and proliferation of individual cells, which builds a local advantage for daughter cells that is cumulative. This has potentially important consequences. In cancers, especially before strongly selective cytotoxic therapy, the assumption that the largest clones must be the cells with deterministic proliferative ability may not always hold true. In development, the gradual loss of clonal diversity as "superstars" take over the population may erode the resilience of the system to somatic mutations, which may have occurred early in clonal growth.

Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system.

The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3ß and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.

Incomplete penetrance: The role of stochasticity in developmental cell colonization.

Cell colonization during embryonic development involves cells migrating and proliferating over growing tissues. Unsuccessful colonization, resulting from genetic causes, can result in various birth defects. However not all individuals with the same mutation show the disease. This is termed incomplete penetrance, and it even extends to discordancy in monozygotic (identical) twins. A one-dimensional agent-based model of cell migration and proliferation within a growing tissue is presented, where the position of every cell is recorded at any time. We develop a new model that approximates this agent-based process - rather than requiring the precise configuration of cells within the tissue, the new model records the total number of cells, the position of the most advanced cell, and then invokes an approximation for how the cells are distributed. The probability mass function (PMF) for the most advanced cell is obtained for both the agent-based model and its approximation. The two PMFs compare extremely well, but using the approximation is computationally faster. Success or failure of colonization is probabilistic. For example for sufficiently high proliferation rate the colonization is assured. However, if the proliferation rate is sufficiently low, there will be a lower, say 50%, chance of success. These results provide insights into the puzzle of incomplete penetrance of a disease phenotype, especially in monozygotic twins. Indeed, stochastic cell behavior (amplified by disease-causing mutations) within the colonization process may play a key role in incomplete penetrance, rather than differences in genes, their expression or environmental conditions.

Parameters affecting efficiency of in ovo electroporation of the avian neural tube and crest.

BACKGROUND: Many variations in avian in ovo transfection of the neural tube/crest have been reported, but never compared quantitatively. RESULTS: Genome integrating pT2K-CAGGS-GFP and pCAGGS-T2TP transposase plasmids were co-electroporated into quail E2 embryo trunk neural tube and the proportion of GFP-expressing neural cells was counted 1 and 7 days later. Electroporation efficiency increased with plasmid concentration and pulse number but plateaued at, respectively, above 1.25 µg/µL and 3 pulses. Bilateral electroporation transfected more cells than unilateral but less than that anticipated by doubling the unilateral treatment. Holding the concentration of GFP plasmid constant and varying the transposase plasmid concentration revealed an optimum ratio of, in this case, 4:1 (1.2 µg/µL:0.3 µg/µL). Leaving transfected embryos to E9 confirmed that expression was maintained in vivo with the transposase system, but declined with non-integrated plasmid. Transfection of neural crest cells was low if electroporated less than 6-8 hr before emigration. We propose this indicates loss of epithelial integrity well prior to exit. We suggest this event be termed epithelio-mesenchymal transition sensu stricto, whereas the term delamination be reserved for the later emigration from the neural epithelium. CONCLUSIONS: Co-electroporation in ovo must take into account plasmid(s) concentration and ratio, pulse number, pulse directionality, and timing.

Simple rules for a "simple" nervous system? Molecular and biomathematical approaches to enteric nervous system formation and malformation.

We review morphogenesis of the enteric nervous system from migratory neural crest cells, and defects of this process such as Hirschsprung disease, centering on cell motility and assembly, and cell adhesion and extracellular matrix molecules, along with cell proliferation and growth factors. We then review continuum and agent-based (cellular automata) models with rules of cell movement and logistical proliferation. Both movement and proliferation at the individual cell level are modeled with stochastic components from which stereotyped outcomes emerge at the population level. These models reproduced the wave-like colonization of the intestine by enteric neural crest cells, and several new properties emerged, such as colonization by frontal expansion, which were later confirmed biologically. These models predict a surprising level of clonal heterogeneity both in terms of number and distribution of daughter cells. Biologically, migrating cells form stable chains made up of unstable cells, but this is not seen in the initial model. We outline additional rules for cell differentiation into neurons, axon extension, cell-axon and cell-cell adhesions, chemotaxis and repulsion which can reproduce chain migration. After the migration stage, the cells re-arrange as a network of ganglia. Changes in cell adhesion molecules parallel this, and we describe additional rules based on Steinberg's Differential Adhesion Hypothesis, reflecting changing levels of adhesion in neural crest cells and neurons. This was able to reproduce enteric ganglionation in a model. Mouse mutants with disturbances of enteric nervous system morphogenesis are discussed, and these suggest future refinement of the models. The modeling suggests a relatively simple set of cell behavioral rules could account for complex patterns of morphogenesis. The model has allowed the proposal that Hirschsprung disease is mostly an enteric neural crest cell proliferation defect, not a defect of cell migration. In addition, the model suggests an explanations for zonal and skip segment variants of Hirschsprung disease, and also gives a novel stochastic explanation for the observed discordancy of Hirschsprung disease in identical twins.

Poly(ethylene glycol)-modified PAMAM-Fe3O4-doxorubicin triads with the potential for improved therapeutic efficacy: generation-dependent increased drug loading and retention at neutral pH and increased release at acidic pH.

Polyamidoamine (PAMAM) dendrimer-coated magnetic nanoparticles are a promising drug-delivery system that can enhance the therapeutic effects of chemotherapy drugs, such as doxorubicin (DOX), with minimized side effects. This work explores the optimization of the potential therapeutic efficiency of PAMAM-Fe3O4-DOX triads. Different generations (G3, G5, and G6) of PAMAMs were synthesized and modified with poly(ethylene glycol) (PEG) and then used to encapsulate glutamic acid-modified Fe3O4 nanoparticles. The Fe3O4-dendrimer carriers (Fe3O4-DGx where x = the generation 3, 5, or 6 of dendrimers) were electrostatically conjugated with drug DOX. The loading and releasing efficiencies of DOX increased with the PAMAM generation from 3 to 6. The loading efficiencies of DOX molecules were 87, 93, and 96% for generations 3, 5, and 6, respectively. At pH 5, the DOX release efficiencies within 24 h were approximately 60, 68, and 80% for generations 3, 5, and 6, respectively. At pH 7.4, the DOX releasing efficiency was as low as ∼ 15%. Compared to the negative control, the PAMAM-Fe3O4-DOX triads showed only mild toxicity against human cervical adenocarcinoma cell line HeLa at pH 7.4, which indicated that DOX can be fairly benignly carried and sparingly released until PAMAM-Fe3O4-DOX is taken up into the cell.

Spatial and temporal dynamics of cell generations within an invasion wave: a link to cell lineage tracing.

Mathematical models of a cell invasion wave have included both continuum partial differential equation (PDE) approaches and discrete agent-based cellular automata (CA) approaches. Here we are interested in modelling the spatial and temporal dynamics of the number of divisions (generation number) that cells have undergone by any time point within an invasion wave. In the CA framework this is performed from agent lineage tracings, while in the PDE approach a multi-species generalized Fisher equation is derived for the cell density within each generation. Both paradigms exhibit qualitatively similar cell generation densities that are spatially organized, with agents of low generation number rapidly attaining a steady state (with average generation number increasing linearly with distance) behind the moving wave and with evolving high generation number at the wavefront. This regularity in the generation spatial distributions is in contrast to the highly stochastic nature of the underlying lineage dynamics of the population. In addition, we construct a method for determining the lineage tracings of all agents without labelling and tracking the agents, but through either a knowledge of the spatial distribution of the generations or the number of agents in each generation. This involves determining generation-dependent proliferation probabilities and using these to define a generation-dependent Galton-Watson (GDGW) process. Monte-Carlo simulations of the GDGW process are used to determine the individual lineage tracings. The lineages of the GDGW process are analyzed using Lorenz curves and found to be similar to outcomes generated by direct lineage tracing in CA realizations. This analysis provides the basis for a potentially useful technique for deducing cell lineage data when imaging every cell is not feasible.

Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells.

INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells. METHODS: MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann-Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P < 0.05). RESULTS: Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity. CONCLUSIONS: This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.

Spatial analysis of multi-species exclusion processes: application to neural crest cell migration in the embryonic gut.

Hindbrain (vagal) neural crest cells become relatively uniformly distributed along the embryonic intestine during the rostral to caudal colonization wave which forms the enteric nervous system (ENS). When vagal neural crest cells are labeled before migration in avian embryos by in ovo electroporation, the distribution of labeled neural crest cells in the ENS varies vastly. In some cases, the labeled neural crest cells appear evenly distributed and interspersed with unlabeled neural crest cells along the entire intestine. However, in most specimens, labeled cells occur in relatively discrete patches of varying position, area, and cell number. To determine reasons for these differences, we use a discrete cellular automata (CA) model incorporating the underlying cellular processes of neural crest cell movement and proliferation on a growing domain, representing the elongation of the intestine during development. We use multi-species CA agents corresponding to labeled and unlabeled neural crest cells. The spatial distributions of the CA agents are quantified in terms of an index. This investigation suggests that (i) the percentage of the initial neural crest cell population that is labeled and (ii) the ratio of cell proliferation to motility are the two key parameters producing the extreme differences in spatial distributions observed in avian embryos.

A Novel Method for Identifying the Transition Zone in Long-Segment Hirschsprung Disease: Investigating the Muscle Unit to Ganglion Ratio.

Hirschsprung disease (HSCR) is characterised by the absence of enteric ganglia along variable lengths of the distal bowel. Current gold standard treatment involves the surgical resection of the defective, aganglionic bowel. Clear and reliable distinction of the normoganglionated bowel from the transition zone is key for successful resection of the entire defective bowel, and the avoidance of subsequent postoperative complications. However, the intraoperative nature of the tissue analysis and the variability of patient samples, sample preparation, and operator objectivity, make reproducible identification of the transition zone difficult. Here, we have described a novel method for using muscle units as a distinctive landmark for quantifying the density of enteric ganglia in resection specimens from HSCR patients. We show that the muscle unit to ganglion ratio is greater in the transition zone when compared with the proximal, normoganglionated region for long-segment HSCR patients. Patients with short-segment HSCR were also investigated, however, the muscle unit to ganglion ratio was not significantly different in these patients. Immunohistochemical examination of individual ganglia showed that there were no differences in the proportions of either enteric neurons or glial cells through the different regions of the resected colon. In addition, we identified that the size of enteric ganglia was smaller for patients that went on to develop HSCR associated enterocolitis; although the density of ganglia, as determined by the muscle unit to ganglia ratio, was not different when compared with patients that had no further complications. This suggests that subtle changes in the enteric nervous system, even in the "normoganglionated" colon, could be involved in changes in immune function and subsequent bacterial dysbiosis.