The technical foundation of GutPath is CITE-seq, a method that simultaneously captures transcriptomes and roughly 100 surface proteins from the same single cells. The researchers profiled the intestinal epithelium, the underlying lamina propria and the mesenteric lymph node in mice infected with segmented filamentous bacteria, Yersinia pseudotuberculosis, the helminth Nippostrongylus brasiliensis, the fungus Candida albicans, the apicomplexan parasite Cryptosporidium parvum and murine norovirus strain CR6. After rigorous quality control, the atlas retained 505,956 high-quality cells, split between roughly 321,000 ileal cells and 185,000 lymph node cells, with individual cells averaging more than 6,000 transcripts. Annotation combined automated reference-based methods with extensive manual curation, resolving 91 distinct transcriptional cell states in the ileum and 49 in the lymph node, organized in multiple layers from broad lineages down to highly specific phenotypes.

A crucial validation step was to confirm that the atlas faithfully reproduces established immunological archetypes. Differential abundance analysis of cell neighborhoods showed that Nippostrongylus infection expanded tuft and goblet cell populations, the classic hyperplasia seen in helminth-driven type 2 immunity, while Yersinia drove a marked recruitment of neutrophils and monocytes, hallmarks of acute bacterial infection. Cryptosporidium and Yersinia both increased Ifng expression in natural killer and natural killer T cells and enriched the interferon-gamma signaling program, consistent with known interferon-dependent control of the parasite. Murine norovirus produced a more modest type I interferon signature, and both Candida colonization and segmented filamentous bacteria enriched T helper 17 differentiation programs in lymph node T cells, with Candida also elevating transforming growth factor-beta signaling and hypoxia-related gene sets. In other words, the atlas captures the canonical immunology of each model, giving researchers confidence in what it reports about the unexpected.

That confidence matters because the most striking findings emerged from the epithelial compartment rather than the immune one. When the team performed unbiased differential expression analysis, the magnitude of transcriptional change varied enormously across infections, with Nippostrongylus and Yersinia producing the most robust responses and murine norovirus and Candida the weakest. At stringent fold-change thresholds, intestinal epithelial cells, including enterocytes, stem cells, transit-amplifying cells and goblet cells, together with fibroblasts, emerged as major drivers of the innate response across all infections. Strikingly, the genes induced in stem cells, transit-amplifying cells and enterocytes showed remarkably little overlap between infections, indicating that the epithelium mounts largely pathogen-specific transcriptional programs rather than a generic alarm response.

To understand these programs, the researchers exploited the fact that their sample preparation captured the full developmental lifespan of the enterocyte, from Lgr5-positive stem cells in the crypts through transit-amplifying cells to mature absorptive cells at the villus tip. Pseudotime trajectory inference, validated against well-established marker genes, allowed them to map gene expression along this differentiation axis. Among 4,278 genes whose expression changes along the trajectory in naive mice, 82 encode solute carrier transporters, the transmembrane proteins on apical and basal enterocyte surfaces that move nutrients and ions. Twenty-nine of these transporters, 35 percent, were dysregulated in at least one infection. Some, such as the nucleoside transporter Slc28a2 and the taurine transporter Slc6a6, retained their developmental timing but were downregulated by Yersinia and Nippostrongylus, while others, including the phosphate symporter Slc20a1 and the mitochondrial glutathione transporter Slc25a39, showed infection-altered timing of expression, suggesting either spatially distinct niches along the villus or a broader shift in enterocyte state.

The helminth model revealed how deeply infection can reach into epithelial metabolism. Scoring metabolic tasks with the scCellfie framework showed that enterocytes from Nippostrongylus-infected mice had the most pronounced alterations of any condition, concentrated in lipid metabolism, with reduced synthesis of secondary bile acids and altered handling of fatty acids such as palmitate, linoleate and arachidonate. Cell-cell communication analysis predicted that these enterocytes were receiving interleukin-4 and interleukin-13 signals, the canonical type 2 cytokines, and were producing fibroblast growth factor family ligands, including Fgf15, a key regulator of bile acid and cholesterol metabolism. Mass spectrometry confirmed the biology: epithelial scrapings, but not luminal contents, showed elevated fatty acids in infected mice, and the accumulation correlated with worm burden, linking pathogen load directly to metabolic disruption of the tissue.

Yersinia pseudotuberculosis produced an even more distinctive epithelial phenotype. Clustering imbalance analysis showed that only Yersinia, among all six models, generated a unique enterocyte transcriptional state, which the authors named Yps enterocytes. Pseudotime modeling revealed that Yersinia elicited more temporally regulated differentially expressed genes than all other infections combined, and the 132 genes defining Yps enterocytes included acute-phase proteins such as Saa1 and Saa2, the antimicrobial effectors Nos2 and Reg3a, and a strong enrichment of immune defense ontology terms. Transcription factor motif analysis pointed to STAT3, a downstream effector of many cytokine cascades, and to SREBF1, a regulator of fatty acid synthesis, with the STAT3 signature being specific to the Yps population.

Spatial transcriptomics then connected this state to tissue pathology. Yersinia infection produces dense inflammatory foci called pyogranulomas, rich in bacteria, neutrophils and monocytes. Using the 10x Genomics Xenium platform with a 5,000-gene panel, the team profiled more than 680,000 spatially resolved cells and computationally unrolled intestinal Swiss rolls into a linear proximal-to-distal axis. Yps enterocyte gene expression peaked precisely at pyogranuloma locations, and cell-cell communication predictions identified an IL-22/STAT3/IL-18 signaling axis: IL-22 expression peaked at the pyogranuloma, STAT3-activated enterocytes produced IL-18, and IL-18 receptor signaling was predicted to reach innate lymphoid cells, natural killer cells and natural killer T cells within the inflammatory focus. Stereo-seq analysis showed that Yersinia transcripts remained confined to the base of the pyogranuloma and did not extend into the overlying enterocytes, and immunohistochemistry confirmed phospho-STAT1 activation at pyogranulomas and broader phospho-STAT3 activation across the tissue, supporting a model in which immune-derived cytokines, rather than direct bacterial contact, drive the epithelial phenotype.

Perhaps the most conceptually important observation is that some infection-induced programs ripple far beyond the sites of heaviest pathogen burden. Genes such as Nos2 and Hif1a were restricted to enterocytes immediately above pyogranulomas, but Saa1, Stat3 and the iron exporter Slc40a1 were elevated across the entire ileum. A parallel ripple appeared in the helminth model, where a proximal intestinal infection remodeled the distal ileum. The authors caution that their atlas captures a single early time point per infection and that fragile granulocytes may be underrepresented, but the resource itself, freely queryable through CELLxGENE at gutpath.org with all raw data and code available, offers the immunology community a reference for label transfer, hypothesis generation and the study of how focal infections and inflammatory lesions, including those of Crohn’s disease, broadcast their effects across the intestinal landscape.

Subject of Research: Single-cell transcriptomic profiling of intestinal epithelial and immune cell responses to diverse enteric infections in mice

Article Title: Diverse infections transcriptionally reprogram the intestinal epithelium and epithelial–immune cell interactions

Article References: Hart, A., Merolle, M., Howard, C., Haskins, B. E., Cohn, I. S., Bobba, S., Xiao, R., Yang, Y., Cadwell, K., Ma, J., Yano, H., Hou, X., Wallbank, B. A., Cutillo, D., Ivanov, I. I., Striepen, B., Shin, S., Brodsky, I. E., Artis, D., … Artis, D. (2026). Diverse infections transcriptionally reprogram the intestinal epithelium and epithelial–immune cell interactions. Nature Immunology. https://doi.org/10.1038/s41590-026-02665-6

Image Credits: AI Generated

DOI: 10.1038/s41590-026-02665-6

Keywords: GutPath, single-cell RNA sequencing, CITE-seq, intestinal epithelium, ileum, enterocytes, Yersinia pseudotuberculosis, Nippostrongylus brasiliensis, Cryptosporidium, mucosal immunology, spatial transcriptomics, cell-cell communication

The answer, according to the study, is a resounding no — at least for the surgery itself. Using genetic mouse models in which HIF1α was specifically deleted from the intestinal epithelium, the researchers demonstrated that the absence of this factor did not diminish the dramatic metabolic improvements normally achieved after bariatric procedures. Mice lacking intestinal HIF1α still experienced the characteristic benefits of the surgery, including reduced body weight, improved glucose tolerance, and favorable changes in fat distribution. In other words, the celebrated metabolic rewiring triggered by bariatric surgery proceeds perfectly well without this oxygen-responsive transcription factor pulling the strings in the gut.

That finding alone would have been notable, but the study’s second act is where the story becomes genuinely intriguing. When the same HIF1α-deficient mice were challenged not with surgery but with a high-fat diet, the protective role of the protein suddenly came into sharp focus. Animals lacking intestinal HIF1α gained significantly more weight on the obesogenic diet than their genetically intact counterparts, and their livers told an equally sobering tale: hepatic steatosis, the abnormal accumulation of fat in liver tissue, developed more readily and more severely. The gut oxygen sensor, it turns out, is not a passive bystander in metabolic disease but an active defender against dietary stress.

This distinction between the two experimental contexts is scientifically meaningful rather than merely academic. Bariatric surgery operates largely through mechanisms independent of ordinary dietary physiology — rapid changes in bile acid signaling, gut hormone secretion, microbiome composition, and nutrient sensing that create a fundamentally altered metabolic environment. Diet-induced obesity, by contrast, unfolds gradually through the slow accumulation of caloric excess and the chronic, low-grade inflammatory and hypoxic stresses it imposes on tissues. HIF1α appears to be critical for withstanding the latter condition while being dispensable for the former, suggesting that the factor functions primarily as a buffer against the physiological consequences of nutrient overload rather than as a mediator of surgical metabolic reprogramming.

To appreciate why the intestine was the logical place to look, it helps to consider the unique biology of gut tissue. The intestinal epithelium sits at the interface between a nutrient-rich lumen and the oxygen-sensitive vasculature of the body, creating a physiological gradient that researchers describe as functional hypoxia. Even in healthy animals, the cells lining the gut experience oxygen levels far lower than most other tissues. HIF1α responds to this environment by activating dozens of target genes involved in barrier integrity, angiogenesis, glycolytic metabolism, and inflammatory regulation. Disrupting this system, the new data indicate, leaves the gut metabolically vulnerable in ways that ripple outward to the whole body, manifesting as increased adiposity and fatty liver disease.

The hepatic connection deserves particular attention. Non-alcoholic fatty liver disease affects roughly a quarter of the global population and represents one of the most serious downstream consequences of obesity, capable of progressing to inflammation, fibrosis, and cirrhosis. If intestinal HIF1α helps protect the liver from fat accumulation, then understanding the signaling pathway between the gut and the liver becomes a matter of substantial clinical relevance. The new findings point toward gut-derived signals — whether barrier-related, microbial, or endocrine — as modulators of hepatic lipid handling, reinforcing a growing body of evidence that liver health begins in the intestine.

Methodologically, the study relied on conditional knockout technology, a cornerstone of modern mouse genetics that allows researchers to remove a gene from a specific tissue while leaving it intact everywhere else. This precision matters enormously for HIF1α, a protein expressed throughout the body with roles ranging from red blood cell production to tumor biology. A whole-body deletion would be lethal or hopelessly confounded; an intestinal epithelium-specific deletion cleanly isolates the gut’s contribution. By comparing knockout and control animals across both surgical and dietary paradigms, the authors could disentangle two biological questions that had previously been tangled together: whether HIF1α transmits the benefits of bariatric surgery, and whether it defends against dietary obesity.

The clinical implications cut in several directions at once. For the millions of patients undergoing bariatric surgery each year, the findings offer reassurance of a negative kind: there is no evidence that natural variation in intestinal HIF1α function would blunt the surgery’s effectiveness. For the far larger population at risk of diet-induced obesity and fatty liver disease, however, the study highlights a potential therapeutic target. If pharmacological activation of intestinal HIF1α — through microbiome modulation, dietary interventions, or drug development — can mimic the protective effect observed in the mouse models, it could open a new avenue for preventing or treating metabolic disease without surgery.

That translational leap will require considerable additional work. Mouse models of obesity and bariatric surgery capture only part of human physiology, and HIF1α is a notoriously pleiotropic factor whose activation can carry risks as well as benefits, including contributions to certain cancers and inflammatory conditions. The researchers themselves are careful to frame the results as a foundation rather than a prescription. Still, the conceptual payoff is clear: the metabolic benefits of bariatric surgery and the body’s natural defenses against dietary obesity travel along partially separate molecular roads, and intestinal HIF1α stands as a guardian on one road but not the other.

As the global burden of obesity and its hepatic complications continues to climb, studies like this one refine the field’s understanding of where interventions can do the most good. Bariatric surgery will remain a powerful tool whose mechanisms are only gradually being mapped. Meanwhile, the humble oxygen sensor in the gut lining — a protein once studied mainly in the context of altitude adaptation and tumor hypoxia — has emerged as an unexpected protector of metabolic health, one whose full therapeutic potential is only beginning to be explored.

Subject of Research: The role of intestinal HIF1α in bariatric surgery outcomes, diet-induced obesity, and hepatic steatosis

Article Title: Intestinal HIF1α is dispensable for bariatric surgery-mediated metabolic benefits but protects against diet-induced obesity and hepatic steatosis

Article References: Cao, C., Liu, Y., Tan, X., Zhao, Y., Jaime, H., Chu, Y., He, M., Hua, R., Yao, Q., & Shao, Y. (2026). Intestinal HIF1α is dispensable for bariatric surgery-mediated metabolic benefits but protects against diet-induced obesity and hepatic steatosis. International Journal of Obesity. https://doi.org/10.1038/s41366-026-02212-1

Image Credits: AI Generated

DOI: 10.1038/s41366-026-02212-1

Keywords: HIF1α, bariatric surgery, diet-induced obesity, hepatic steatosis, intestinal epithelium, hypoxia signaling, metabolic disease, fatty liver, gut-liver axis, glucose tolerance, mouse models, International Journal of Obesity

BEST4⁺ cells are defined by a distinctive combination of genes, including BEST4, OTOP2, CA7 and GUCY2C. In the small intestine, they also show particularly high expression of CFTR, the chloride and bicarbonate channel best known for its role in cystic fibrosis. Together, these markers suggest a specialized role in moving negatively charged ions across the epithelium. Chloride and bicarbonate transport draw water into or out of the intestinal lumen and help regulate the chemical environment surrounding epithelial cells and mucus. OTOP2 adds a sensing capability: this proton-selective channel can respond to changes in extracellular acidity. CA7 may support intracellular bicarbonate production, while GUCY2C encodes guanylyl cyclase C, a receptor that converts extracellular signals into cyclic GMP. The resulting molecular profile is unusually coherent for a relatively small cell population.

The cells were first associated with the intestinal epithelium through studies of bestrophin expression, but modern single-cell sequencing made their identity much clearer. These analyses separate individual epithelial cells according to their RNA profiles, allowing researchers to identify populations that conventional staining can overlook. BEST4⁺ cells appear early in human intestinal development, reportedly as early as gestational week 11, and generally represent less than 5 percent of the fetal epithelial compartment. In adults, their abundance varies by region, with reported enrichment in parts of the jejunum, ileum and colon. Small-intestinal cells are concentrated toward the upper and middle villus, whereas colonic cells tend to occupy apical crypt regions. The regional differences suggest that a shared core program may be adapted to local pH, microbial exposure, mucus and transport demands.

The developmental identity of BEST4⁺ cells is still being debated. Several lines of evidence place them near the end of an absorptive differentiation pathway. They occupy post-mitotic compartments, lack conventional proliferation markers and express genes associated with mature enterocytes and colonocytes, including VIL1, AQP8 and SLC26A3. Human organoid experiments indicate that NOTCH signaling is required for their emergence, and the transcription factor SPIB appears indispensable: removing SPIB with CRISPR-based gene editing prevented BEST4⁺ cell generation even when NOTCH signaling remained active. Yet other findings point toward connections with the secretory lineage. Trajectory analyses in human tissue have identified a low-probability link to ATOH1-positive secretory progenitors, while zebrafish lineage-tracing experiments suggest that related cells can arise from secretory precursors. These differences may reflect species, anatomical region or inflammatory state rather than a single universal developmental route.

Under normal conditions, BEST4⁺ cells may act as coordinated pH and ion-transport units. BEST4 belongs to the bestrophin family of calcium-activated anion channels, which can conduct chloride and bicarbonate when intracellular calcium rises. CFTR provides another major route for apical anion secretion. The cells also express guanylin and uroguanylin, the endogenous ligands for GUCY2C, alongside the receptor itself. This arrangement could create an autocrine circuit in which locally produced ligands stimulate cyclic GMP, activate downstream protein kinases and increase CFTR activity. Bicarbonate secretion is important beyond fluid balance: it helps neutralize acidity near the epithelial surface and allows newly released MUC2 mucin to hydrate and expand into an effective protective layer. The review therefore proposes that BEST4⁺ cells may support mucus-barrier assembly in cooperation with goblet cells, although direct proof that these cells provide the critical bicarbonate flux is still lacking.

The same machinery can be exploited during secretory diarrhea. Bacterial heat-stable enterotoxin, or STa, binds and activates GUCY2C, raising intracellular cyclic GMP and stimulating CFTR through protein kinase G. Cholera toxin and the heat-labile toxin of enterotoxigenic Escherichia coli activate adenylate cyclase through persistent cyclic AMP signaling, leading to protein kinase A-mediated CFTR activation. In both cases, excessive chloride and bicarbonate secretion promotes water movement into the lumen. The review highlights evidence that BEST4⁺ cells are unusually equipped for this response because they co-express GUCY2C and CFTR at functionally relevant levels. Human intestinal organoids exposed to these pathways swell as fluid accumulates. Investigational inhibitors of GUCY2C or CFTR can reduce secretion in experimental systems, but broad suppression carries risks because basal GUCY2C signaling also contributes to barrier integrity, mucus hydration and epithelial maintenance.

Viral diarrhea may involve BEST4⁺ cells more indirectly. Porcine epidemic diarrhea virus preferentially damages villus absorptive enterocytes and can impair NHE3, a sodium-hydrogen exchanger needed for sodium-coupled water absorption. Studies in infected piglets have reported reduced expression of several water and nutrient transporters, together with increased ileal CFTR transcripts. Electrical measurements of infected jejunal tissue also indicate enhanced secretory responses. These observations are consistent with a shift away from absorption and toward secretion, and the presence of CFTR-rich BEST4⁺ cells makes them plausible contributors. But the available evidence does not show that the virus infects BEST4⁺ cells or that the cells are responsible for the altered current. Rotavirus offers another possible route: its NSP4 protein disturbs calcium signaling and triggers ADP-dependent calcium waves in neighboring uninfected cells. BEST4⁺ cells could respond as bystander effectors through calcium-sensitive anion channels, but this remains a testable hypothesis.

Norovirus likewise causes changes that could intersect with the BEST4⁺ program, including reduced epithelial resistance and increased electrogenic chloride secretion. The virus can replicate in differentiated enterocytes and some enteroendocrine cells, but there is no direct evidence that mature BEST4⁺ cells support norovirus replication. Inflammation may nevertheless alter their numbers or activity. In human organoids, interferon-gamma increases BEST4⁺ cell differentiation through a SPIB-dependent mechanism, and the resulting cells show stronger CFTR-dependent secretion after toxin exposure. If antiviral inflammation produces a similar response in living intestine, it could amplify fluid loss during acute disease. Conversely, bicarbonate secretion and mucus hydration might aid barrier repair during recovery. The review also connects BEST4⁺ cells to inflammatory bowel disease, where their abundance and expression of transport and metal-buffering genes appear altered, and to cystic fibrosis, in which defective CFTR trafficking may leave these high-CFTR cells unable to regulate anion transport, luminal acidity and mucus hydration.

These possibilities make BEST4⁺ cells attractive but challenging therapeutic targets. A drug that blocks pathological GUCY2C or CFTR activation could reduce fluid loss, yet permanent or systemic inhibition might undermine normal mucosal defense. Experimental CFTR inhibitors have reduced toxin-induced secretion in rodents, although some show limited solubility, rapid washout, narrow dosing windows or off-target effects on mitochondria. A more selective strategy may involve ADRA2A, an adrenergic receptor enriched in human BEST4⁺ cells; activating it suppresses cyclic AMP secretion and reverses cholera-toxin-induced swelling in enriched organoids. Such findings remain preclinical. The next steps will require lineage-specific genetic tools, direct electrophysiological measurements and disease experiments in animals that actually possess a conserved BEST4⁺ population. Conventional laboratory mice lack a canonical intestinal Best4 lineage, making rats, pigs, zebrafish and human organoids complementary rather than interchangeable models. Until researchers can manipulate these cells in vivo, BEST4⁺ cells should be viewed not as a confirmed master switch for viral diarrhea, but as a promising framework for understanding how infection, inflammation and epithelial ion transport converge.

At the molecular level, the proposed hub function depends on the way several transport systems are colocated rather than on BEST4 alone. Bestrophin channels are described as pentameric calcium-sensitive anion channels with a calcium-binding region, a hydrophobic gate and a cytoplasmic regulatory segment. This architecture provides a potential link between intracellular calcium signals and rapid changes in chloride or bicarbonate permeability. In a BEST4⁺ cell, such calcium-dependent conductance could complement CFTR, whose activity is controlled primarily through cyclic-nucleotide signaling. The two routes therefore offer distinct but potentially convergent means of regulating apical anion movement, while OTOP2 and CA7 could help couple that transport activity to the chemical conditions at the epithelial surface.

That arrangement also helps explain why anatomical location matters. BEST4⁺ cells are reported in the proximal small intestine and at the colonic surface, but their associated transport programs are not identical in every region or species. Villus-associated small-intestinal cells encounter different nutrient, acid and fluid gradients from cells near colonic crypt openings. Cross-species conservation supports a shared cellular program, yet conservation of marker genes does not establish conservation of net ion flux. Differences in epithelial architecture, microbiota, mucus organization and channel abundance could alter the physiological contribution of the same transcriptional cell type. Functional comparisons will therefore need to measure transport in defined regions rather than treating all BEST4⁺ cells as equivalent.

A central experimental challenge is separating correlation from cell-specific causation. High BEST4, GUCY2C or CFTR expression identifies a candidate effector population, but whole-organoid swelling, tissue short-circuit current and bulk transporter measurements integrate responses from many epithelial cells. Stronger tests would combine selective deletion or activation of BEST4⁺ cells with live measurements of intracellular pH, calcium, cyclic GMP, bicarbonate flux and mucus expansion. These experiments could determine whether BEST4 itself is the principal anion pathway, whether it mainly amplifies CFTR-mediated secretion, or whether its greatest contribution is sensing and coordinating responses among neighboring cells. They could also clarify whether toxin-induced secretion requires the endogenous guanylin–uroguanylin circuit or is driven predominantly by pharmacological stimulation of GUCY2C.

Therapeutic development will depend on preserving the distinction between pathological hypersecretion and protective basal transport. GUCY2C signaling and bicarbonate movement may contribute to epithelial maintenance and mucus function even while excessive cyclic GMP or cyclic AMP drives diarrhea. This argues for approaches that limit abnormal signal amplitude, duration or cellular targeting instead of eliminating the pathway entirely. Cell-type-resolved physiology, supported by species with a conserved BEST4⁺ population, should help identify that therapeutic window and establish whether the proposed hub is a druggable controller or primarily a useful map of interacting intestinal transport mechanisms.

Subject of Research: BEST4⁺ intestinal epithelial cells and their role in ion transport and diarrheal mechanisms

Article Title: BEST4⁺ cells: a potential hub of intestinal ion transport and diarrhea manipulation

Article References: Qu, H.-Z., & Wang, X.-Q. (2026). BEST4⁺ cells: a potential hub of intestinal ion transport and diarrhea manipulation. Advanced Biotechnology, 4(3), Article 33. https://doi.org/10.1007/s44307-026-00126-7

Image Credits: AI Generated

DOI: 10.1007/s44307-026-00126-7

Keywords: BEST4⁺ cells, intestinal epithelium, ion transport, CFTR, GC-C signaling, secretory diarrhea, viral gastroenteritis, mucus barrier, BEST4, cells, potential, intestinal