Rab7-dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis.

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier, coupled with microbial dysbiosis, has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability, and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7-dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

Staphylococcus aureus Biofilm-Secreted Factors Cause Mucosal Damage, Mast Cell Infiltration, and Goblet Cell Hyperplasia in a Rat Rhinosinusitis Model.

Chronic rhinosinusitis (CRS) is an inflammatory condition of the sinonasal mucosa. Despite being a common health issue, the exact cause of CRS is yet to be understood. However, research suggests that Staphylococcus aureus, particularly in its biofilm form, is associated with the disease. This study aimed to investigate the impact of long-term exposure to secreted factors of Staphylococcus aureus biofilm (SABSFs), harvested from clinical isolates of non-CRS carrier and CRS patients, on the nasal mucosa in a rat model. Animals were randomised (n = 5/group) to receive daily intranasal instillations of 40 µL (200 µg/µL) SABSFs for 28 days or vehicle control. The sinonasal samples were analysed through histopathology and transcriptome profiling. The results showed that all three intervention groups displayed significant lymphocytic infiltration (p ≤ 0.05). However, only the SABSFs collected from the CRSwNP patient caused significant mucosal damage, mast cell infiltration, and goblet cell hyperplasia compared to the control. The transcriptomics results indicated that SABSFs significantly enriched multiple inflammatory pathways and showed distinct transcriptional expression differences between the control group and the SABSFs collected from CRS patients (p ≤ 0.05). Additionally, the SABSF challenges induced the expression of IgA and IgG but not IgE. This in vivo study indicates that long-term exposure to SABSFs leads to an inflammatory response in the nasal mucosa with increased severity for S. aureus isolated from a CRSwNP patient. Moreover, exposure to SABSFs does not induce local production of IgE.

Intergenerational protective anti-gut commensal immunoglobulin G originates in early life.

Maternal immunoglobulins of the class G (IgGs) protect offspring from enteric infection, but when, where, and how these antibodies are physiologically generated and confer protection remains enigmatic. We found that circulating IgGs in adult mice preferentially bind early-life gut commensal bacteria over their own adult gut commensal bacteria. IgG-secreting plasma cells specific for early-life gut bacteria appear in the intestine soon after weaning, where they remain into adulthood. Manipulating exposure to gut bacteria or plasma cell development before, but not after, weaning reduced IgG-secreting plasma cells targeting early-life gut bacteria throughout life. Further, the development of this anti-gut commensal IgG response coincides with the early-life interval in which goblet cell-associated antigen passages (GAPs) are present in the colon. Offspring of dams "perturbed" by B cell ablation or reduced bacterial exposure in early life were more susceptible to enteric pathogen challenge. In contrast to current concepts, protective maternal IgGs targeted translocating gut commensals in the offspring, not the enteric pathogen. These early-life events affecting anti-commensal IgG production have intergenerational effects for protection of the offspring.

Sex drives colonic mucin sialylation in wild mice.

Mucin protein glycosylation is important in determining biological properties of mucus gels, which form protective barriers at mucosal surfaces of the body such as the intestine. Ecological factors including: age, sex, and diet can change mucus barrier properties by modulating mucin glycosylation. However, as our understanding stems from controlled laboratory studies in house mice, the combined influence of ecological factors on mucin glycosylation in real-world contexts remains limited. In this study, we used histological staining with 'Alcian Blue, Periodic Acid, Schiff's' and 'High-Iron diamine' to assess the acidic nature of mucins stored within goblet cells of the intestine, in a wild mouse population (Mus musculus). Using statistical models, we identified sex as among the most influential ecological factors determining the acidity of intestinal mucin glycans in wild mice. Our data from wild mice and experiments using laboratory mice suggest estrogen signalling associates with an increase in the relative abundance of sialylated mucins. Thus, estrogen signalling may underpin sex differences observed in the colonic mucus of wild and laboratory mice. These findings highlight the significant influence of ecological parameters on mucosal barrier sites and the complementary role of wild populations in augmenting standard laboratory studies in the advancement of mucus biology.

Comparing the effect of benzalkonium chloride-preserved, polyquad-preserved, and preservative-free prostaglandin analogue eye drops on cultured human conjunctival goblet cells

Purpose To investigate the effect of benzalkonium chloride (BAK)-preserved latanoprost and bimatoprost, polyquad (PQ)-preserved travoprost, and preservative-free (PF) latanoprost and tafluprost, all prostaglandin analogues (PGAs), on human conjunctival goblet cell (GC) survival. Furthermore, to investigate the effect of BAK-preserved and PF latanoprost on the cytokine secretion from GC. Methods Primary human conjunctival GCs were cultivated from donor tissue. Lactate dehydrogenase (LDH) and tetrazolium dye colorimetric (MTT) assays were used for the assessment of GC survival. A cytometric bead array was employed for measuring secretion of interleukin (IL)-6 and IL-8 from GC. Results BAK-preserved latanoprost and bimatoprost reduced cell survival by 28% (p = 0.0133) and 20% (p = 0.0208), respectively, in the LDH assay compared to a negative control. BAK-preserved latanoprost reduced cell proliferation by 54% (p = 0.003), BAK-preserved bimatoprost by 45% (p = 0.006), PQ-preserved travoprost by 16% (p = 0.0041), and PF latanoprost by 19% (p = 0.0001), in the MTT assay compared to a negative control. Only PF tafluprost did not affect the GCs in either assay. BAK-preserved latanoprost caused an increase in the secretion of pro-inflammatory IL-6 and IL-8 (p = 0.0001 and p = 0.0019, respectively) compared to a negative control, which PF latanoprost did not. Conclusion BAK-preserved PGA eye drops were more cytotoxic to GCs than PQ-preserved and PF PGA eye drops. BAK-preserved latanoprost induced an inflammatory response in GC. Treatment with PF and PQ-preserved PGA eye drops could mean better tolerability and adherence in glaucoma patients compared to treatment with BAK-preserved PGA eye drops. (AU)

Macro- and micro morphology of the olfactory organ of African bonytongue, Heterotis niloticus (Cuvier 1829), compared with other species of the family Osteoglossidae (Teleostei).

Osteoglossiformes (bonytongue fishes) possess many morphological specializations associated with functions such as airbreathing, feeding, and electroreception. The olfactory organ also varies among species, notably in the family Osteoglossidae. Herein, we describe the olfactory organ of an osteoglossid, Heterotis niloticus, to compare it with the olfactory organs of other osteoglossiforms. We demonstrate the presence of an olfactory rosette within the olfactory chamber. This structure consists of a short median raphe surrounded by olfactory lamellae, which possess dorsal lamellar processes. On the surface of the olfactory lamellae, there are secondary lamellae formed by the olfactory epithelium. Within the olfactory epithelium, two zones can be distinguished: parallel brands of sensory cells located in the cavities between the secondary lamellae and a nonsensory area covering the remaining part of the olfactory lamellae. The olfactory epithelium is formed by ciliated and microvillus olfactory sensory neurons, supporting cells, goblet cells, basal cells and ciliated nonsensory cells. Additionally, rodlet cells were observed. The results confirm large variability in terms of the olfactory organ of Osteoglossiformes, particularly of Osteoglossidae, and support the secondary lamellae evolution hypothesis within this family.

SLE serum induces altered goblet cell differentiation and leakiness in human intestinal organoids.

Human intestinal epithelial cells are the interface between luminal content and basally residing immune cells. They form a tight monolayer that constantly secretes mucus creating a multilayered protective barrier. Alterations in this barrier can lead to increased permeability which is common in systemic lupus erythematosus (SLE) patients. However, it remains unexplored how the barrier is affected. Here, we present an in vitro model specifically designed to examine the effects of SLE on epithelial cells. We utilize human colon organoids that are stimulated with serum from SLE patients. Combining transcriptomic with functional analyses revealed that SLE serum induced an expression profile marked by a reduction of goblet cell markers and changed mucus composition. In addition, organoids exhibited imbalanced cellular composition along with enhanced permeability, altered mitochondrial function, and an interferon gene signature. Similarly, transcriptomic analysis of SLE colon biopsies revealed a downregulation of secretory markers. Our work uncovers a crucial connection between SLE and intestinal homeostasis that might be promoted in vivo through the blood, offering insights into the causal connection of barrier dysfunction and autoimmune diseases.

Extraction of Mucins from the Mammalian Intestinal Tract.

In the intestine, mucus covering the mucosa plays a critical role in maintaining gut homeostasis by protecting the mucosa from invasion by commensal bacteria. The gut mucus is composed primarily of MUC2 mucin secreted by goblet cells. MUC2 is highly O-glycosylated, and O-glycans are necessary for the function and polymer structure of MUC2. In addition, recent evidence revealed that several glycan modifications, such as sialylation and sulfation, confer resistance of mucins to proteolysis and affect the viscosity and lubricity of mucus. Therefore, characterizing glycan structures of mucins is required to understand their functions fully. In this chapter, we describe how to purify secreted mucins from the mammalian intestine for analysis of their glycan structures. This description includes the extraction of MUC2 mucin from the mucosal surface of the mouse colon and colon explants.

Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection.

Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.

Human conjunctiva organoids to study ocular surface homeostasis and disease.

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

Human apical-out nasal organoids reveal an essential role of matrix metalloproteinases in airway epithelial differentiation.

Extracellular matrix (ECM) assembly/disassembly is a critical regulator for airway epithelial development and remodeling. Airway organoid is widely used in respiratory research, yet there is limited study to indicate the roles and mechanisms of ECM organization in epithelial growth and differentiation by using in vitro organoid system. Moreover, most of current Matrigel-based airway organoids are in basal-out orientation where accessing the apical surface is challenging. We present a human apical-out airway organoid using a biochemically defined hybrid hydrogel system. During human nasal epithelial progenitor cells (hNEPCs) differentiation, the gel gradually degrade, leading to the organoid apical surfaces facing outward. The expression and activity of ECM-degrading enzymes, matrix metalloproteinases (MMP7, MMP9, MMP10 and MMP13) increases during organoid differentiation, where inhibition of MMPs significantly suppresses the normal ciliation, resulting in increased goblet cell proportion. Moreover, a decrease of MMPs is found in goblet cell hyperplastic epithelium in inflammatory mucosa. This system reveals essential roles of epithelial-derived MMPs on epithelial cell fate determination, and provides an applicable platform enabling further study for ECM in regulating airway development in health and diseases.

Activation of goblet-cell stress sensor IRE1ß is controlled by the mucin chaperone AGR2.

Intestinal goblet cells are secretory cells specialized in the production of mucins, and as such are challenged by the need for efficient protein folding. Goblet cells express Inositol-Requiring Enzyme-1ß (IRE1ß), a unique sensor in the unfolded protein response (UPR), which is part of an adaptive mechanism that regulates the demands of mucin production and secretion. However, how IRE1ß activity is tuned to mucus folding load remains unknown. We identified the disulfide isomerase and mucin chaperone AGR2 as a goblet cell-specific protein that crucially regulates IRE1ß-, but not IRE1α-mediated signaling. AGR2 binding to IRE1ß disrupts IRE1ß oligomerization, thereby blocking its downstream endonuclease activity. Depletion of endogenous AGR2 from goblet cells induces spontaneous IRE1ß activation, suggesting that alterations in AGR2 availability in the endoplasmic reticulum set the threshold for IRE1ß activation. We found that AGR2 mutants lacking their catalytic cysteine, or displaying the disease-associated mutation H117Y, were no longer able to dampen IRE1ß activity. Collectively, these results demonstrate that AGR2 is a central chaperone regulating the goblet cell UPR by acting as a rheostat of IRE1ß endonuclease activity.

The IRE1ß-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells.

Effector mechanisms of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) are well-characterised, but how ER proteostasis is sensed is less well understood. Here, we exploited the beta isoform of the UPR transducer IRE1, that is specific to mucin-producing cells in order to gauge the relative regulatory roles of activating ligands and repressing chaperones of the specialised ER of goblet cells. Replacement of the stress-sensing luminal domain of endogenous IRE1α in CHO cells (normally expressing neither mucin nor IRE1ß) with the luminal domain of IRE1ß deregulated basal IRE1 activity. The mucin-specific chaperone AGR2 repressed IRE1 activity in cells expressing the domain-swapped IRE1ß/α chimera, but had no effect on IRE1α. Introduction of the goblet cell-specific client MUC2 reversed AGR2-mediated repression of the IRE1ß/α chimera. In vitro, AGR2 actively de-stabilised the IRE1ß luminal domain dimer and formed a reversible complex with the inactive monomer. These features of the IRE1ß-AGR2 couple suggest that active repression of IRE1ß by a specialised mucin chaperone subordinates IRE1 activity to a proteostatic challenge unique to goblet cells, a challenge that is otherwise poorly recognised by the pervasive UPR transducers.

Sphingosine kinase 1-specific inhibitor PF543 reduces goblet cell metaplasia of bronchial epithelium in an acute asthma model.

Sphingosine kinase 1 (SPHK1) has been shown to play a key role in the pathogenesis of asthma where SPHK1-generated sphingosine-1-phosphate (S1P) is known to mediate innate and adaptive immunity while promoting mast cell degranulation. Goblet cell metaplasia (GCM) contributes to airway obstruction in asthma and has been demonstrated in animal models. We investigated the role of PF543, a SPHK1-specific inhibitor, in preventing the pathogenesis of GCM using a murine (C57BL/6) model of allergen-induced acute asthma. Treatment with PF543 before triple allergen exposure (DRA: House dust mite, Ragweed pollen, and Aspergillus) reduced inflammation, eosinophilic response, and GCM followed by reduced airway hyperreactivity to intravenous methacholine. Furthermore, DRA exposure was associated with increased expression of SPHK1 in the airway epithelium which was reduced by PF543. DRA-induced reduction of acetylated α-tubulin in airway epithelium was associated with an increased expression of NOTCH2 and SPDEF which was prevented by PF543. In vitro studies using human primary airway epithelial cells showed that inhibition of SPHK1 using PF543 prevented an allergen-induced increase of both NOTCH2 and SPDEF. siRNA silencing of SPHK1 prevented the allergen-induced increase of both NOTCH2 and SPDEF. NOTCH2 silencing was associated with a reduction of SPDEF but not that of SPHK1 upon allergen exposure. Our studies demonstrate that inhibition of SPHK1 protected allergen-challenged airways by preventing GCM and airway hyperreactivity, associated with downregulation of the NOTCH2-SPDEF signaling pathway. This suggests a potential novel link between SPHK1, GCM, and airway remodeling in asthma.NEW & NOTEWORTHY The role of SPHK1-specific inhibitor, PF543, in preventing goblet cell metaplasia (GCM) and airway hyperreactivity (AHR) is established in an allergen-induced mouse model. This protection was associated with the downregulation of NOTCH2-SPDEF signaling pathway, suggesting a novel link between SPHK1, GCM, and AHR.

Surfactant protein D prevents mucin overproduction in airway goblet cells via SIRPα.

Mucin overproduction is a common feature of chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), and exacerbates their underlying respiratory condition. Surfactant protein D (SP-D) protects against airway diseases through modulation of immune reactions, but whether it also exerts direct effects on airway epithelial cells has remained unclear. Therefore, we sought to investigate the inhibitory role of SP-D on mucin production in airway epithelial cells. We prepared air-liquid interface (ALI) cultures of human primary bronchial epithelial cells (HBECs), which recapitulated a well-differentiated human airway epithelium. Benzo(a)pyrene (BaP), a key toxicant in cigarette smoke, induced mucin 5AC (MUC5AC) production in ALI-cultured HBECs, airway secretory cell lines, and airway epithelia of mice. Then, the protective effects of SP-D against the BaP-induced mucin overproduction were examined. BaP increased MUC5AC production in ALI cultures of HBECs, and this effect was attenuated by SP-D. SP-D also suppressed the BaP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and MUC5AC expression in NCI-H292 goblet-like cells, but not in NCI-H441 club-like cells. Signal regulatory protein α (SIRPα) was found to be expressed in HBECs and NCI-H292 cells but absent in NCI-H441 cells. In NCI-H292 cells, SP-D activated SH2 domain-containing tyrosine phosphatase-1 (SHP-1), downstream of SIRPα, and knockdown of SIRPα abolished the suppressive effects of SP-D on BaP-induced ERK phosphorylation and MUC5AC production. Consistent with these in vitro findings, intratracheal instillation of SP-D prevented the BaP-induced phosphorylation of ERK and Muc5ac expression in airway epithelial cells in a mouse model. SP-D acts directly on airway epithelial cells to inhibit mucin secretion through ligation of SIRPα and SHP-1-mediated dephosphorylation of ERK. Targeting of SIRPα is therefore a potential new therapeutic approach to suppression of mucin hypersecretion in chronic airway diseases such as COPD and asthma.

Reduced miR-513a-5p expression in COPD may regulate airway mucous cell hyperplasia through TFR1-dependent signaling.

Airway mucous cell metaplasia and mucous hypersecretion is one of the key characteristic pathophysiological status of chronic obstructive pulmonary disease (COPD). micro(mi)RNAs are acknowledged as non-encoding RNA molecules playing important roles in gene expression regulation. In this study, we searched the Gene Expression Omnibus (GEO) database for the differentially expressed miRNAs between COPD and non-COPD controls with bioinformatics analysis. Finally, we focused on miR-513a-5p and investigated the potential mechanism by which miR-513a-5p regulates airway mucous hypersecretion and goblet cell metaplasia. A dual-luciferase reporter assay was then showing that miR-513a-5p targeted the 3'-UTR of TFR1 and inhibited its expression in vitro. In vivo transfection demonstrated that TFR1 downregulation partially blocked MUC5AC hypersecretion and goblet cell hyperplasia in COPD model rats. In vitro study, CSE increased the intracellular expression and secretion of MUC5AC by BEAS-2B branchial epithelial cells in the BEAS-2B cell and THP-1 cell coculture system. Coculture with either miR-513a-5p mimic-pretreated or TFR1-deficient THP-1 cells attenuated intracellular MUC5AC expression in BEAS-2B cells exposed to CSE. ELISA demonstrated that transfection of TFR1 siRNA or pretreatment with miR-513a-5p mimic reduced the secretion of inflammatory factors that are responsible for airway goblet cell hyperplasia, such as IL-1ß, IL-13, and IL-17, by THP-1 cells after CSE stimulation. Our findings supported that miR-513a-5p/TFR1 signaling axis might activate macrophages as well as promote airway inflammation and airway mucous cell hyperplasia in COPD.

Comparing the effect of benzalkonium chloride-preserved, polyquad-preserved, and preservative-free prostaglandin analogue eye drops on cultured human conjunctival goblet cells.

PURPOSE: To investigate the effect of benzalkonium chloride (BAK)-preserved latanoprost and bimatoprost, polyquad (PQ)-preserved travoprost, and preservative-free (PF) latanoprost and tafluprost, all prostaglandin analogues (PGAs), on human conjunctival goblet cell (GC) survival. Furthermore, to investigate the effect of BAK-preserved and PF latanoprost on the cytokine secretion from GC. METHODS: Primary human conjunctival GCs were cultivated from donor tissue. Lactate dehydrogenase (LDH) and tetrazolium dye colorimetric (MTT) assays were used for the assessment of GC survival. A cytometric bead array was employed for measuring secretion of interleukin (IL)-6 and IL-8 from GC. RESULTS: BAK-preserved latanoprost and bimatoprost reduced cell survival by 28% (p = 0.0133) and 20% (p = 0.0208), respectively, in the LDH assay compared to a negative control. BAK-preserved latanoprost reduced cell proliferation by 54% (p = 0.003), BAK-preserved bimatoprost by 45% (p = 0.006), PQ-preserved travoprost by 16% (p = 0.0041), and PF latanoprost by 19% (p = 0.0001), in the MTT assay compared to a negative control. Only PF tafluprost did not affect the GCs in either assay. BAK-preserved latanoprost caused an increase in the secretion of pro-inflammatory IL-6 and IL-8 (p = 0.0001 and p = 0.0019, respectively) compared to a negative control, which PF latanoprost did not. CONCLUSION: BAK-preserved PGA eye drops were more cytotoxic to GCs than PQ-preserved and PF PGA eye drops. BAK-preserved latanoprost induced an inflammatory response in GC. Treatment with PF and PQ-preserved PGA eye drops could mean better tolerability and adherence in glaucoma patients compared to treatment with BAK-preserved PGA eye drops.

Macauba (Acrocomia aculeata) pulp oil has the potential to enhance the intestinal barrier morphology, goblet cell proliferation and gut microbiota composition in mice fed a high-fat diet.

Macauba (Acrocomia aculeata) is a palm tree native from Brazil, whose pulp is rich in oil that has a high content of oleic acid and carotenoids. Macauba pulp oil can bring health benefits due to its bioactive compounds; however, its effects on gut health are unknown. Thus, the objective of this study was to evaluate the effect of macauba pulp oil on the intestinal health in mice fed a high-fat (HF) diet. Male C57BL1/6 mice were randomly divided into three groups (10 animals/group): control diet, HF diet and HF diet with 4 % of macauba pulp oil (HFM). Concentration of short-chain fatty acids (SCFA), faecal pH and histomorphometric analysis of the colon were performed. Content of colon samples was used on microbiome analysis using 16S rRNA amplicon sequencing. Animals from the HFM group had higher butyric acid content and goblet cells number, greater circular and longitudinal muscle layer and higher α-diversity compared with the HF group. Moreover, consumption of MPO reduced Desulfobacterota phylum, Ruminococcaceae, Oscillospiraceae, Prevotellaceae, Bifidobacteriaceae family, Faecalibacterium, Prevotella, Ruminococcus and Enterorhabdus genus. Therefore, macauba pulp oil was able to modulate the gut microbiota and enhance intestinal barrier morphology, showing preventive effects on gut dysbiosis in mice fed a HF diet.