Porcine intestinal goblet cells: Guardians of mucosal immunity and barrier function

Abstract

Goblet cells are a type of secretory cell in the intestinal epithelium, characterized by their continuous production and secretion of mucin. Goblet cell-associated antigen passage (GAP)-mediated antigen presentation offers intestinal tolerance to innocuous luminal antigens and systemic resistance to infection. However, multiple factors can impair goblet cell abundance influencing mucin production in swine. Therefore, strategies aimed at maintaining porcine goblet cell homeostasis could provide an important role in the protection of gut health, ultimately enhancing porcine health and production performance. This review summarizes the current knowledge on porcine goblet cells, including methods of examination, their development and functions, and demonstrations of the presence of GAPs in the porcine small intestine. This review also lists the factors and nutrients that influence goblet cell homeostasis in swine production and discusses the potential roles of porcine goblet cells in nutrient digestion and absorption. Overall, this review highlights the importance of preserving the goblet cell-mediated portion of intestinal homeostasis and advances the understanding of porcine goblet cells, providing opportunities for the development of new methods to improve gut health in swine production.

1. Introduction

Gut health has become a key research focus in the swine industry due to its profound impact on production performance and overall pig health (Ali et al., 2022). The gut is not only responsible for the efficient digestion and absorption of dietary nutrients, but also functions as a vital immune organ which harbors over 70% of bodily immune cells (Blikslager et al., 2007). However, various factors such as stress, pathogens, and mycotoxins can compromise porcine gut health, leading to intestinal disorders such as diarrhea and subsequent declines in production performance (Recharla et al., 2022; Wang et al., 2025). Therefore, improving gut health is a crucial strategy for raising economic benefits and ensuring the sustainable development in modern swine production.

The monolayer intestinal epithelium is composed of absorptive epithelial cells (such as enterocytes) and secretory epithelial cells (such as goblet cells, Paneth cells, and enteroendocrine cells), which play vital roles in nutrient absorption and mucosal immunity (Beumer and Clevers, 2021). Over the past decade, intestinal goblet cells have garnered increasing attention due to their essential involvement in intestinal health regulation during both homeostasis and disease states such as ulcerative colitis and Crohn's disease (Allaire et al., 2018). Enterocytes are the most abundant cell type in intestinal villi, while goblet cells account for approximately 5% of all villus cells (Modina et al., 2021). The density of goblet cells progressively increases from the duodenum to the colon (Gustafsson and Johansson, 2022). Notably, porcine goblet cells are abundant in both villi and crypts of the small intestine, and goblet cells show high homology among humans, pigs, and mice (Wiarda et al., 2023). As key components of intestinal innate immunity, goblet cells synthesize and secrete mucins (MUCs) to form a epithelium-covered mucus layer, thereby preventing the excessive exposure to microorganisms and food antigens in intestinal lumen (Gustafsson and Johansson, 2022). Interestingly, a recent study has revealed that goblet cells also contribute to systemic immune protection through sampling luminal antigens and delivering them to lamina propria immune cells (Udayan et al., 2025). Consequently, targeting goblet cell homeostasis could represent a potential strategy for improving intestinal health. In contrast to human and murine goblet cells, porcine goblet cells have received less attention in swine production research. To advance the understanding of porcine goblet cells, this review discusses the current knowledge of porcine goblet cells and summarizes potential influencing factors and nutritional regulation of porcine goblet cells in swine production.

2. Porcine goblet cells and MUCs

In porcine goblet cells, the apical portion is filled with MUC-containing secretory vesicles, and the bottom portion is occupied by nuclei (Kalita et al., 2021). Goblet cell-derived MUCs are rich in serine, threonine, and proline (Cheng et al., 2025), suggesting the great necessity of these amino acids to MUC homeostasis. The vital roles of MUCs in maintaining intestinal homeostasis and gut health have been comprehensively discussed by Cheng et al. (2025). There are various functional goblet cell subpopulations in the gut of humans and mice, such as canonical goblet cell, non-canonical goblet cell, and defence profile goblet cell (Gustafsson and Johansson, 2022). Whether similar goblet cell subpopulations exist in the porcine gut remains unclear.

Transcriptomic analysis of exfoliated epithelial cells in the porcine descending colon demonstrated the general presence of goblet cell markers in exfoliated contents which implies the rapid renewal of porcine goblet cells (Yoon et al., 2021). The rapid turnover of porcine goblet cells may be associated with the substantial turnover of mucus in pigs (Modina et al., 2021). The microstructure of porcine small intestinal mucus resembles that of humans in terms of particle penetrability (Krupa et al., 2020). It has been estimated that 55-kg pigs can produce 3.9 kg MUCs per kg feed intake (Moran and Bedford, 2023). The entire contents of villus goblet cells are continuously secreted and replenished within approximately 12 h (Schneider et al., 2018).

In addition to MUCs, porcine goblet cells possess multiple functional proteins such as intelectin, aquaporin 9 (AQP9), and chloride channel accessory 1 (CLCA1) in their secretory vesicles (Keeler et al., 2022; Ren et al., 2023; Wrackmeyer et al., 2006). Intelectin is a Ca²⁺-dependent and D-galactosyl-specific lectin and plays a vital role in the intestinal innate immune response to parasite infection (Wrackmeyer et al., 2006). Aquaporin 9 is a small transmembrane protein supporting water transport across membranes (Ren et al., 2023), which could be important for water homeostasis in porcine goblet cells. Several studies have demonstrated the regulatory role of AQP9 in inflammatory signaling pathways in humans and mice (De Santis et al., 2018; Takeuchi et al., 2018). Chloride channel accessory 1 is required for mucin 5AC (MUC5AC) expression and mucus production in the porcine gut, and CLCA1 knockout pigs exhibit significant decreases in goblet cell number in the jejunum, ileum, and colon (Keeler et al., 2022).

3. Materials and methods for porcine goblet cell examination

Multiple materials and methods can be used to examine alterations in goblet cells. In experiments involving pigs, fresh intestinal tissue or mucosa could be collected for goblet cell examination. For in vitro treatment with drugs or nutrients, porcine intestinal explants or organoids are appropriate materials to observe goblet cells. In intestinal organoids, goblet cell enrichment (up to 80% of all cells) can be achieved by treatments with specific drugs (Basak et al., 2017). However, intestinal explants are not suitable for long-time treatment due to their short lifespan. Additionally, intestinal porcine epithelial cell line-J2 (IPEC-J2) cells could also be potential material for goblet cell examination due to the successful immunostaining of MUC2, a well-known goblet cell marker. To study goblet cell functions, gene knockout of atonal homolog 1 (Atoh1), a key regulator for secretory epithelial cell differentiation, could be conducted to ablate goblet cells in mice, though it also leads to deletion of Paneth cells and enteroendocrine cells. It is important to note the inherent limitations of the above in vitro models. While containing multiple epithelial cell types, the porcine intestinal organoid model lacks key components of the in vivo microenvironment such as lamina propria immune cells, enteric nerves, vasculature, and microbiota. Similarly, the IPEC-J2 monolayer is an epithelial cell model without immune or microbial interactions, and the intestinal explant model ignores the potential impact of intestinal microbiota. Therefore, goblet cell-associated findings derived from these models should be interpreted within their specific contexts and validated in more complex in vivo settings.

Goblet cells are characterized by the abundant MUC-containing secretory vesicles in the upper part of cytoplasm. Therefore, the examination of goblet cells is generally associated with their MUCs and secretory vesicles. Several methods such as histochemical staining and transmission electron microscope (TEM) can be utilized to visualize the abundant secretory vesicles in the cytoplasm of porcine goblet cells. After hematoxylin and eosin (H&E) staining, vacuoles in villi and crypts can be preliminarily used to identify potential porcine goblet cells. However, for accurate identification and quantification, specific staining methods for MUCs or goblet cell markers are required. In contrast to H&E staining, alcian blue-periodic acid Schiff (AB-PAS) staining and high iron diamine (HID)-AB staining are more intuitive to observe porcine goblet cells. Alcian blue-periodic acid Schiff staining can distinguish neutral MUCs and acidic MUCs, and HID-AB staining can identify sulfomucins and sialomucins. Notably, Rieger et al. (2019) have established a modified goblet cell staining method to observe neutral MUCs, sulfomucins, and sialomucins on one tissue section. Goblet cells could also be visualized by staining with lectins such as ulex europaeus agglutinin I (UEA1), wheat germ agglutinin (WGA), and maackia amurensis lectin II (MALII). Ulex europaeus agglutinin I staining are more obvious in small intestinal villi of unweaned piglets and small intestinal crypts of weaned piglets (Brown et al., 1991). Using TEM can examine the changes of intracellular components, including secretory vesicles and mitochondria, in porcine goblet cells. In addition to secretory vesicle visualization, it is also convenient to examine porcine goblet cells by assessing goblet cell markers such as MUC2 and trefoil factor family 3 (TFF3) via qPCR, Western blot, and immunostaining. Notably, goblet cells possess abundant endoplasmic reticulum (ER) to synthesize proteins (Gustafsson and Johansson, 2022), which means that they are highly susceptible to ER stress that blocks protein synthesis. Thus, it is of great necessity to investigate goblet cell markers at both gene and protein levels to reveal the actual changes within goblet cells.

To accurately examine the changes in goblet cells, goblet cells could be isolated from the small intestine or colon using flow cytometry and cell sorting. In mice, goblet cells are identified as CD45^- CD24^- CK-18⁺ UEA-1⁺, and enterocytes are identified as CD45^- CD24^- CK-18^- UEA-1^- (Udayan et al., 2025). However, whether this sorting mode applies to porcine goblet cells remains unclear, highlighting an urgent need for a porcine-specific goblet cell sorting strategy. Validating surface-binding lectins (such as UEA1) or discovering novel surface markers via single-cell RNA sequencing could facilitate the establishment of a reliable method to isolate porcine goblet cells, which is crucial for advancing porcine goblet cell studies.

To observe mucus thickness on tissue section, the harvested tissue must undergo additional processing such as fixation with Carnoy's solution and liquid nitrogen cryopreservation (Roehe et al., 2018; Tang et al., 2022). In addition, McCright et al. (2022) have established an in vitro gut model with co-culture of Caco-2 cells and HT-29-MTX cells to study the biophysical properties of porcine small intestinal mucus. However, it is important to note that while this human cell-based model is valuable for studying the physical attributes of porcine mucus, it lacks the species-specific cellular and molecular context of the porcine intestine. Therefore, its applications are primarily confined to investigations of porcine mucus biophysics, and findings derived from this model should be carefully extrapolated to porcine intestinal biology.

4. Development of goblet cells in porcine small intestine

In both humans and pigs, goblet cells emerge in the small intestine before birth (Gustafsson and Johansson, 2022). By d 40 of gestation, differentiated goblet cells are detected in the duodenum of fetal pigs. By d 45 to 90 of gestation, the number of immature goblet cells is dramatically increased in the duodenum and jejunum of fetal pigs. By d 110 of gestation, porcine goblet cells tend to be mature with an obvious goblet shape and abundant secretory vesicles (Fig. 1) (Dekaney et al., 1997). The prenatal development of porcine goblet cells provides solid protection against outside pathogenic microorganisms after birth.

Fig. 1.

Porcine goblet cell development. Porcine goblet cells are differentiated from intestinal stem cells at E40. During E45-E90, the number of immature goblet cells is increasing. At E110, most goblet cells are mature. In pre-weaning period after birth, goblet cell number is gradually increasing. Within 14 d after weaning, porcine goblet cell number exhibits an increasing-decreasing-increasing change due to dietary transition and physiological development. E = embryonic day; P = postnatal day.

Postnatal goblet cell development is regulated by both growth factors (such as epidermal growth factor [EGF] in breast milk) and environmental stimulation (Gustafsson and Johansson, 2022). In mice, the number of goblet cells is gradually elevated within 2 weeks after birth (Gustafsson and Johansson, 2022). Compared with neonatal piglets, 7-d-old sucking piglets display no changes in goblet cell number in the jejunum, while goblet cell number in intestinal organoids from 7-d-old piglets is slightly increased compared to that from neonatal piglets (Yin et al., 2022). When compared to 7- or 14-d-old piglets, 18-d-old piglets possess more sulfomucin-containing goblet cells in duodenum and ileum (Fig. 1) (Brown et al., 2006). These findings suggest the relatively insensitive responses of porcine goblet cells to growth factors and environmental stimulation. The role of intestinal microbiota in postnatal goblet cell development could be controversial. Germ-free neonatal piglets subjected to 13-d fecal microbiota transplantation (FMT) with adult porcine fecal suspension exhibited fewer goblet cells in the proximal small intestine (Shirkey et al., 2006). Meanwhile, conventionally raised piglets undergoing 10-d FMT showed decreased diarrhea rates and elevated goblet cell numbers and MUC2 expression in the ileum and colon (Hu et al., 2018).

There are various types of cells in the intestinal epithelium monolayer, indicating that epithelial cell differentiation is a highly-regulated and intricate biological process. Intestinal epithelial cell differentiation is modulated by multiple signaling pathways such as Notch signaling and Wnt/β-catenin signaling (Beumer and Clevers, 2021), with Goblet cell differentiation being promoted by the inactivation of both pathways. Lineage commitment towards absorptive cells or secretory cells is dependent on the activity of Notch signaling. Dibenzazepine, an inhibitor of Notch signaling, increases intestinal Krüppel-like factor 4 (Klf4) expression and facilitates goblet cell differentiation (Ghaleb et al., 2008). Hairy and enhancer of split 1 (Hes1), a target gene of Notch signaling, can inhibit the transcription of Atoh1 to block secretory cell commitment (VanDussen et al., 2012). Atonal homolog 1 is a target gene of Wnt/β-catenin signaling, and its knockout results in the loss of secretory epithelial cells including goblet cells (Yang et al., 2001).

5. Multifaceted functions of porcine goblet cells

5.1. Mucin production and secretion

Mucin biosynthesis in goblet cells is a complex process, and MUC2 is the best-studied MUC. Mucin 2 biosynthesis is started in the ER where MUC2 is initially formed through folding, dimerization and initial N-glycosylation (Gustafsson and Johansson, 2022). Mucin 2 dimers are then transferred to the Golgi apparatus and are extensively O-glycosylated. After O-glycosylation, MUC2 is packed in secretory vesicles with other functional proteins under low pH and high calcium conditions (Ambort et al., 2012). Mucin-containing secretory vesicles are released through exocytosis which can be rapidly triggered by acetylcholine-mediated goblet cell muscarinic acetylcholine receptor 1 (mAChR1) activation (Gustafsson et al., 2021). Secretory vesicles could also be affected by many factors such as diet and microbial signals (Sittipo et al., 2019). For instance, protein-free diets lead to a significant decrease in MUC production (Lien et al., 2001).

O-glycosylated MUCs are the primary structural components of intestinal mucus. The MUC glycan chain length could be important for intestinal health. In diarrheal piglets, short glycans are abundant while longer glycans are deficient in the colon (Xia et al., 2022). According to O-glycan types, goblet cell-derived MUCs can be divided into neutral MUCs and acidic MUCs that includes sulfomucins and sialomucins. Acidic MUCs (primarily sulfomucins) exhibit strong resistance to bacterial enzymatic degradation, protecting the intestinal barrier (Fontaine et al., 1998). In piglets, sialomucin-containing goblet cells are located in small intestinal crypts, and sulfomucin-containing goblet cells mainly reside in small intestinal villi and colonic crypts (Xia et al., 2021). Diarrheal piglets display increased number of neutral MUC-containing goblet cells and decreased number of acidic MUC-containing goblet cells (Xia et al., 2022), suggesting the importance of acidic MUCs in intestinal health. The above alterations to O-glycan levels in MUCs could play an important role in porcine diarrhea. As mentioned earlier, the lack of acidic MUCs could facilitate bacterial invasion and impair the gut barrier due to their resistance to bacterial enzymatic degradation (Fontaine et al., 1998). Furthermore, MUC O-glycans can enhance short chain fatty acid (SCFA) production to promote gut barrier homeostasis (Hino et al., 2020), and fermentation of short MUC O-glycans from diarrheal piglets by the gut microbiota result in significant decreases to levels of SCFAs (Xia et al., 2022), compromising gut barrier homeostasis. Additionally, fermentation of short MUC O-glycans from diarrheal piglets boosts Lactobacillus abundance in the colon of diarrheal piglets (Xia et al., 2022). The increase in Lactobacillus-associated lactate in the colon of diarrheal piglets could be a key trigger of diarrhea since the absorption of lactate is very slow (Tsukahara and Ushida, 2001). Lactate accumulation in the porcine colon leads to an osmotic load causing water secretion from the gut mucosa (Hoshi et al., 1994). Thus, short MUC O-glycans could be a cause of porcine diarrhea due to their capacity to decrease SCFA production and increase Lactobacillus and lactate abundance.

5.2. Goblet cell-associated antigen passage (GAP)-mediated antigen presentation

In addition to providing MUC, goblet cells have also been shown to deliver luminal antigens to immune cells in the lamina propria via GAPs. In 2012, Newberry and his colleagues from Washington University School of Medicine discovered an unexpected antigen presentation pathway termed GAP in humans and mice (McDole et al., 2012). In the steady state, goblet cells can deliver low molecular weight soluble antigens from the intestinal lumen to lamina propria CD103⁺CX₃CR1^- dendritic cells that induce regulatory T cell development and promote immunoglobulin A (IgA) and interleukin (IL)-10 production, thus maintaining intestinal tolerance to innocuous luminal antigens (McDole et al., 2012; Kulkarni et al., 2020). Goblet cell-associated antigen passage formation can be rapidly induced by acetylcholine-mediated muscarinic acetylcholine receptor 3/4 (mAChR3/4) activation in goblet cells (Gustafsson et al., 2021).

Before weaning, GAPs are only formed in colonic goblet cells, and small intestinal GAPs are repressed by breast milk EGF (Knoop et al., 2017). It has been recently reported that colonic GAP-mediated translocation of live Lactobacillus animalis to mesenteric lymph nodes (MLN) and the spleen provides solid protection against systemic Escherichia coli infection in the preweaning individual (Udayan et al., 2025). In the healthy adult individual, GAPs are only formed in small intestinal goblet cells, and colonic GAPs are inhibited by goblet cell-intrinsic myeloid differentiation primary response 88 (Myd88)-dependent microbial sensing and the subsequent activation of the EGF receptor (EGFR) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways (Knoop et al., 2015). Notably, colonic goblet cells express higher levels of Toll-like receptor (TLR) 1, TLR2, TLR4, and TLR5 than small intestinal goblet cells, which renders colonic goblet cells more susceptible to microbial signals (Knoop et al., 2015). However, in the condition of Salmonella infection, Salmonella utilizes small intestinal GAPs to translocate to MLNs (Kulkarni et al., 2018). Salmonella-induced IL-1β production robustly inhibits small intestinal GAPs to impair antigen-specific T cell proliferation (Kulkarni et al., 2018), which could be a self-protection response to Salmonella infection.

Goblet cell-associated antigen passages could be of great importance to porcine health due to their essential involvement in intestinal immune surveillance and tolerance, and systemic protection. However, in swine production, little is known about intestinal GAPs. To investigate the presence of GAPs in pigs, 10 kD rhodamine-conjugated dextran was injected into small intestinal lumen of healthy 50-d-old pigs. As expected, the existence of GAPs in the intestinal epithelium (white asterisk) was demonstrated by the staining of dextran and MUC2 in one cell (Fig. 2A). Dextran is also ingested by non-goblet cells in the epithelium as shown by the lack of MUC2 staining in dextran-positive cells (white arrow). Indeed, other secretory cells such as enteroendocrine cells and Paneth cells can absorb luminal antigens, despite cell-mediated antigen presentation being less common in other cells when compared to goblet cell-mediated antigen presentation (Kulkarni et al., 2020). Additionally, the different phases of GAP-mediated antigen presentation were also observed (Fig. 2B). Goblet cell ingested dextran is mainly concentrated in the apical portion (phase I), before being translocated to the bottom portion (phase II). After ingestion, dextran in goblet cells is delivered to lamina propria immune cells adjacent to goblet cells (phase III). These findings successfully demonstrate the presence of GAPs in the small intestine of pigs, providing a fundamental basis for the further research on GAP-mediated intestinal health in swine production. In sucking piglets, mature goblet cells do not express EGFR (Schweiger et al., 2003). Therefore, it could be speculated that small intestinal GAPs in sucking piglets will not be inhibited by breast milk EGF, which may be explored in future work.

Fig. 2.

The presence of GAPs in porcine small intestine. Ten kD rhodamine-conjugated dextran (D1824, Thermo Fisher Scientific Inc., Carlsbad, CA, USA) was injected into small intestinal lumen of healthy 50-d-old growing pigs. After 1 h, pigs were painlessly sacrificed for sample collection. (A) GAPs and non-goblet cell-mediated antigen presentation were observed by dextran (red) and MUC2 (green) staining in porcine small intestine. Scale bars: 50 μm (left) and 20 μm (middle and right). (B) The different phases of GAPs in porcine small intestine. Goblet cell ingested dextran is mainly concentrated in the apical portion (phase I), before being translocated to the bottom portion (phase II). After ingestion, dextran in goblet cells is delivered to lamina propria immune cells adjacent to goblet cells (phase III). Scale bar: 10 μm. GAPs = goblet cell-associated antigen passages; MUC2 = mucin 2.

6. Influencing factors on goblet cell homeostasis during swine production

Although goblet cells play an important role in maintaining intestinal homeostasis, they are susceptible to many factors during swine production, such as weaning, growth retardation, infection, mycotoxin, antibiotic, and stress (Fig. 3).

Fig. 3.

The influencing factors of goblet cell homeostasis during swine production mainly include six aspects: weaning, growth retardation, infection, mycotoxin, antibiotic, and stress. IUGR = intrauterine growth restriction.

6.1. Weaning

From birth to weaning, ileal villus height and the small intestinal goblet cell number are increased in piglets (Saleem et al., 2023; Yuan et al., 2021). However, during the weaning period, gut health, including goblet cell homeostasis, in piglets is disturbed due to the dietary transition from breast milk to feeds (Lerch et al., 2022). Over the first 14 d after weaning, the villus height shows a decreasing trend before increasing again, and the crypt depth gradually increases (Wang et al., 2022), suggesting early gut barrier impairment induced by weaning. On d 1 after weaning, the number of goblet cells is transiently elevated in the jejunum and ileum of piglets (Fig. 1), indicating the potential of goblet cells to restore gut homeostasis in response to weaning stress (Wang et al., 2022). On d 3 to 7 after weaning, the number of porcine goblet cells returns to pre-weaning level (Wang et al., 2022). On d 14 after weaning, significant increases in the number of goblet cells and goblet cell marker gene expression emerges in the whole small intestine (Fig. 1) (Wang et al., 2022), which could be attributed to the physiological development of porcine goblet cells. Weaning additionally affects the chemical modification of MUCs. On d 5 after weaning, the number of sialomucin-containing goblet cells is decreased and the number of sulfomucin-containing goblet cells is increased in the small intestinal crypts of piglets (Brown et al., 1988). On d 13 after weaning, sialomucin-containing goblet cells are predominant in small intestinal crypts again (Brown et al., 1988).

In addition to dietary transition, weaning age also has great impact on porcine goblet cell homeostasis. Compared with piglets weaned at 21-d-old, piglets weaned at 7- or 14-d-old exhibit impaired intestinal barriers and lowered goblet cell numbers in the ileum and colon (Garcia et al., 2016; Han et al., 2022). These results emphasize the importance of an appropriate weaning age for piglets in to preserve goblet cell-associated gut homeostasis.

Therefore, the weaning-associated fluctuation in goblet cell abundance and MUC composition may temporarily compromise the defense against enteric pathogens, leading to the high incidence of post-weaning diarrhea. Nutritional interventions that support goblet cell recovery during this period (e.g., functional amino acids, bioactive plant extracts) could help stabilize gut barrier function and improve resistance to diseases.

6.2. Growth retardation

Intrauterine growth restriction (IUGR) piglets exhibit disrupted growth and development during pregnancy, leading to a significant decrease in fetal birth weight and survival rate (Van Ginneken et al., 2023). The number of Goblet cells in the jejunum and ileum is decreased in newborn IUGR piglets when compared to normal birth weight piglets (Dong et al., 2014). The levels of cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in small intestine are lower in IUGR neonatal piglets than in normal birth weight piglets, which could be attributed to a decreased immune cell number and represent a partially defective innate immune system (Dong et al., 2014). Intrauterine growth restriction-induced defects in porcine goblet cell number and differentiation and MUC expression are still present in sucking stage (14-d-old) (Olszewski et al., 2021) and growing stage (49- and 90-d-old) (Tao et al., 2019; Wang et al., 2024a), suggesting the chronic impairment of intestinal innate immunity in IUGR pigs. Notably, the mRNA expression of delta-like ligand 1 (Dll1), a Notch ligand, is up-regulated in the ileum of IUGR piglets (Zhang et al., 2019), which could result in porcine goblet cell defects since Dll1-mediated Notch signaling activation inhibits goblet cell proliferation (Pellegrinet et al., 2011). In addition to IUGR, premature delivery can also lead to growth retardation in piglets. At birth, the number of small intestinal goblet cells in premature piglets is no different to normal piglets at birth, but a significant decrease of goblet cells in 11-d-old premature piglets is observed compared with 11-d-old normal piglets (Ren et al., 2018).

6.3. Infection

In swine production, piglets are susceptible to intestinal infections due to their immature intestinal immune system. E. coli infection is the primary trigger of diarrhea in piglets. Receptors for E. coli are expressed in small intestinal goblet cells in newborn piglets (Dean and Isaacson, 1985), suggesting that goblet cells in piglets could be affected by E. coli. However, the effects of E. coli on porcine goblet cells are inconsistent. On one hand, E. coli (isolate number UI-VDL 05-27242) infection results in increases to the number and size of goblet cells after 5 d post-infection (Almeida et al., 2013). In germ-free piglets, E. coli O149:K88ac infection increases MUC1 protein expression in the colon (Toth et al., 2023). On the other hand, several studies have reported that E. coli K88 and E. coli W25K infection down-regulates goblet cell number in the whole small intestine and colon in piglets after 4 or 14 d post-infection (Lee et al., 2017; Xu et al., 2023). This controversy could be attributed to the use of different E. coli and the condition of intestinal commensal bacteria. As a robust toxin of E. coli, lipopolysaccharide (LPS) treatment via intraperitoneal injection or dietary supplementation also induces goblet cell loss in the porcine small intestine (Wang et al., 2024b; Zapata et al., 2015). Salmonella typhimurium is another pathogenic bacterium widely existing in swine production, and 6-12-week-old pigs are susceptible to S. typhimurium infection (Won et al., 2021). At 10 min post-infection, S. typhimurium invades porcine small intestinal goblet cells, leading to focal apical disruptions in goblet cells (Meyerholz et al., 2002). S. typhimurium infection also lowers goblet cell number in both the jejunum and colon in piglets (Splichal et al., 2023; Won et al., 2021). In the porcine small intestine, infection of Lawsonia intracellularis, a pathogen causing porcine proliferative enteropathy, leads to decreases in goblet cell number and MUC2 expression which is correlated to the percentage of L. intracellularis-invaded intestinal crypts (Bengtsson et al., 2015; McOrist et al., 1996). L. intracellularis-associated porcine goblet cell defects could be ascribed to weakened Wnt/β-catenin signaling activity and Atoh1 expression (Huan et al., 2017). During the early infection of Brachyspira hyodysenteriae, a pathogen causing swine dysentery, marked deletion of goblet cell MUCs and alteration of MUC glycosylation are observed in the base of porcine colonic crypts (Jacobson et al., 2007; Lin et al., 2023).

Porcine epidemic diarrhea virus (PEDV) infection results in vomiting, diarrhea, and even death in piglets. Porcine epidemic diarrhea virus can invade porcine goblet cells after infection, as shown by positive PEDV-N staining around MUC-containing secretory vesicles, which lowers goblet cell number and MUC2 expression in piglets (Fan et al., 2023; Jung and Saif, 2017). In response to PEDV invasion, porcine goblet cells produce calpain-1, a calcium-activated cysteine protease, to alleviate PEDV-induced intestinal injury (Li et al., 2022). Goblet cell-derived calpain-1 represses PEDV invasion by binding to and hydrolyzing the S1 domain of the viral spike protein (Li et al., 2022). Transmissible gastroenteritis virus (TGEV) also causes serious intestinal diarrhea in piglets. Transmissible gastroenteritis virus invades porcine goblet cells through recognizing a MUC-type glycoprotein in a sialic acid-dependent manner (Schwegmann-Wessels et al., 2003). In the porcine intestinal organoid monolayer, MUC2 protein expression is raised at 24 h and before declining at 48 h after TGEV infection (Yang et al., 2022). However, in the jejunum of piglets, TGEV suppresses Notch signaling activity, promoting goblet cell differentiation, and elevating goblet cell number and MUC2 expression (Wu et al., 2020). During porcine reproductive and respiratory syndrome virus (PRRSV) infection, the levels of neutral MUCs and acidic MUCs in jejunal crypts, as well as MUC2 expression, are dramatically up-regulated in piglets (Zhao et al., 2021), which could be a protective response to PRRSV infection. Porcine goblet cell loss and decreased MUC2 expression are also witnessed in infections by other viruses such as porcine deltacoronavirus (PDCoV) (Zhang et al., 2023), porcine circovirus type 2 (Zlotowski et al., 2009), and porcine rotavirus (Mao et al., 2015). Porcine deltacoronavirus infection activates Notch signaling activity and Hes1 expression blocking goblet cell differentiation in the porcine jejunum, while Notch inhibition mitigates PDCoV-induced goblet cell loss in porcine intestinal organoid monolayer (Zhang et al., 2023).

Compared to infections by pathogenic bacteria and viruses, parasitic infection tends to mildly stimulate porcine goblet cells to induce protective responses. In Ascaris suum infection, porcine goblet cell proliferation is activated at the site of A. suum entry in cecum (Masure et al., 2013). Trichuris suis infection contributes to the increases in goblet cell number and abundances of neutral MUC, acidic MUC, and sulfomucin in porcine colon and cecum (Myhill et al., 2018; Thomsen et al., 2006).

Taken together, these findings highlight the importance of goblet cell integrity and function, which could be targeted as a strategic approach to enhance host resistance against specific pathogens.

6.4. Mycotoxin

Mycotoxins are secondary metabolites of multiple fungi and are highly prevalent in porcine feeds, and mycotoxin ingestion is extremely hazardous to the production performance of pigs (Munoz-Solano et al., 2024; Liu et al., 2025). Deoxynivalenol (DON) is one of the most frequently occurring mycotoxins in porcine feeds. In piglets, DON intake leads to a decrease in goblet cell number in the jejunal and ileal villi rather than crypts (Martinez et al., 2019). Mechanistically, DON activates protein kinase R and the mitogen-activated protein kinase p38 signaling pathways to restrain the expression of resistin-like molecule β, thus down-regulating the expression of MUC1-3 and TFF3 (Graziani et al., 2019; Pinton et al., 2015). As another relevant mycotoxin, fumonisin B1 (FB1) has no effect on porcine goblet cell homeostasis in both the small intestine and small intestinal explants (Bracarense et al., 2012; da Silva et al., 2019). Notably, a DON and FB1-combined challenge further lowers the number of goblet cells when compared to a single DON treatment in porcine jejunal explants (Basso et al., 2013). Deoxynivalenol can also aggravate the loss of jejunal goblet cells in piglets infected with PEDV (Liu et al., 2022). Zearalenone challenge can reduce goblet cell number and TFF3 expression and undermine epithelial barrier in the porcine cecum partly through repressing transforming growth factor-β1 (TGF-β1)/Smads signaling (Zhang et al., 2021). Similarly, other mycotoxins such as patulin and T-2 toxin can also impair goblet cell homeostasis in swine production (Maidana et al., 2016).

6.5. Antibiotic

Antibiotics play a crucial role in modern swine production, primarily employed for the prophylaxis and treatment of bacterial diseases to maintain health and production performance (Sung et al., 2025). The effects of antibiotics on porcine goblet cell homeostasis are diverse. Porcine goblet cell development is advanced by multiple antibiotics such as amoxicillin, fosfomycin, ampicillin, gentamycin, metronidazole, and avilamycin (Jensen et al., 2014; Manzanilla et al., 2006; Martinez et al., 2019; Thymann et al., 2007). However, tunicamycin treatment has been shown to reduce goblet cell number in the jejunum of piglets, which is associated with the induction of ER stress (Chen et al., 2022).

6.6. Stress

In swine production, heat stress dramatically impairs animal welfare and production performance (Guo et al., 2018). When compared to thermoneutral conditions, 3-h heat stress together with 3-h cooling treatment reduces ileal goblet cell number in gilts (Kpodo et al., 2020). In the colon of growing pigs, 7-d heat stress contributes to lowered goblet cell number and intestinal microbiota disorders, and heat stress-mediated porcine intestinal microbiota reduces murine colonic goblet cell number after FMT. In contrast, another study has demonstrated that growing pigs exposed to continuous periods of heat stress (a minimum of 6 h and a maximum of 72 h) exhibit an increased goblet cell number and a reduced villus height/crypt depth ratio in the whole small intestine (Liu et al., 2024b). In addition to heat stress, restraint stress activates corticotropin-releasing hormone receptor 1 signaling, reducing goblet cell number and MUC2 expression in the whole small intestine and colon from pregnant pigs (Xu et al., 2021).

6.7. Other influencing factors

Goblet cell homeostasis varies among different swine breeds. Compared with Landrace piglets, Saba piglets, a unique local pig species in central Yunnan Province, possess a higher number of jejunal goblet cells in a way which is positively correlated with taurodeoxycholic acid (He et al., 2023). When compared with Duroc × Landrace × Yorkshire neonatal piglets, Meishan neonatal piglets exhibit a higher number of goblet cells as well as increased MUC2 expression in the duodenum and jejunum (Dong et al., 2021). Additionally, feed intake has an impact on goblet cell homeostasis in pigs. There are more goblet cells in the cecum of high-feed efficiency pigs (He et al., 2019). In piglets with low feed intake during the initial 3 d after weaning, the size of acidic MUC-containing goblet cells is significantly reduced compared to piglets with high feed intake (Faba et al., 2024). Similarly, dietary restriction also decreases goblet cell number in small intestine of nursing piglets (Nunez et al., 1996). Due to the immature digestive system, soybean antigen protein glycinin and β-conglycinin decrease the number of goblet cells in piglets, which could be attributed to enhanced ER stress and blocked autophagic flux (Wang et al., 2023). Drinking alkaline mineral water facilitates brain-microbe-gut axis-mediated activation of intestinal Wnt/β-catenin signaling to promote the differentiation of secretory cells, including goblet cells, thus preventing diarrhea in piglets (Chen et al., 2023).

7. Porcine goblet cell regulation by nutrients

At present, there are many studies on the effects of nutrients on goblet cell homeostasis in swine production. These nutrients include amino acids, lipids, plant extracts, probiotics, vitamins, elements, and others (Table 1). However, for most nutrients, their underlying mechanisms of influence are largely unknown, prompting further exploration.

Table 1.

Porcine goblet cell regulation by diverse nutrients.

Nutrient category	Nutrient names	Goblet cell homeostasis	References
Amino acid	Threonine	↑	Swiech et al. (2011)
	Glycine	↑	Fan et al. (2019)
	Glutamate	↑	Tan et al. (2019); Kyoung et al. (2021)
	Glutamine	↑	Xing et al. (2017)
	Arginine	↑	Wu et al. (2010)
	Methionine	↑	Li et al. (2014)
Lipid	Phosphatidylethanolamine	↑	Wang et al. (2024a)
	Sodium butyrate	↑	Manzanilla et al. (2006); Sadurni et al. (2023)
	Glycerol monolaurate	↑	Cui et al. (2020)
	Tributyrin	↓	Tugnoli et al. (2020)
	Crude glycerin	↓	Oliveira et al. (2014)
Plant extract	Dihydroartemisinin	↑	Niu et al. (2024)
	Beet pulp	↑	Diao et al. (2020)
	Curcumin	↑	Xun et al. (2015)
	Cynara scolymus extract	↑	Martinez et al. (2019)
	Olive pomace extract	↑	Martinez et al. (2024)
	Cinnamaldehyde	↑	Zhao et al. (2024)
	Carvacrol	↑	Zhao et al. (2024)
	Alginate oligosaccharide	↑	Wan et al. (2018)
	Xylo-oligosaccharides	↑	Chen et al. (2021)
	Fructo-oligosaccharides	↑	Xu et al. (2005)
	Inulin	↑	Xia et al. (2021)
	Pectin	↑	Yin et al. (2024)
Probiotic	Lactobacillus rhamnosus	↑	Deng et al. (2022); Montoya et al. (2024)
	Lactobacillus reuteri	↑	Wang et al. (2020)
	Lactobacillus plantarum	↑	Guerra-Ordaz et al. (2014)
	Lactobacillus brevis	↑	Davis et al. (2007)
	Bacillus amyloliquefaciens	↑	Li et al. (2018); Wang et al. (2024b)
	Bacillus subtilis	↑	He et al. (2020)
	Bacillus pumilus	↑	He et al. (2020)
	Bacillus cereus	↑	Reiter et al. (2006)
	Bifidobacterium animalis	↑	Pang et al. (2022)
	Saccharomyces cerevisiae	↑	Garcia et al. (2019)
Vitamin	Vitamin A	↑	Zhou et al. (2021)
	Vitamin B5	↑	Wang et al. (2024c)
Element	Iron	↑	Deng et al. (2021)
	Zinc	↑	Deka et al. (2014)
	Copper	↑	Deka et al. (2014)
	Selenium	↑	Zheng et al. (2021)
Bile acid	Chenodeoxycholic acid	↑	Song et al. (2021)
Acidifier	Benzoic acid	↑	Liu et al. (2024a)

Both a high protein diet and a low protein diet can increase the number of ileal goblet cells in piglets (Yin et al., 2020, 2021). When compared to a high protein diet, a low protein diet is more beneficial to piglets as evidenced by higher levels of goblet cell abundance and MUC sialylation under the conditions of homeostasis and E. coli infection (Lee et al., 2022; Opapeju et al., 2015). Several amino acids have been demonstrated to improve porcine goblet cell homeostasis, such as threonine, glutamate, and glycine. Among them, threonine is the most important as goblet cell-derived MUCs are rich in threonine (Swiech et al., 2011). Dietary threonine supplementation contributes to marked increases in the number of acidic and neutral MUC-containing goblet cells in the ileal villi and crypts of growing pigs (Swiech et al., 2011). Threonine deficiency in neonatal piglets induces loss of acidic MUC-producing goblet cells and serious diarrhea (Law et al., 2007). Excessive or insufficient levels of true ileal digestible threonine can lead to decreases in MUC expression and sulfomucin abundance in piglets (Wang et al., 2007, 2010). In IUGR piglets, threonine treatment down-regulates Dll1 and Hes1 expression and up-regulates growth factor independent 1 transcription repressor (Gfi1) and Klf4 expression, thereby boosting the number of ileal goblet cells and MUC2 expression (Zhang et al., 2019). Additionally, preweaning supplementation of glycine enhances porcine goblet cell number partly through attenuating ER stress-induced apoptosis in goblet cells (Fan et al., 2019).

The effects of different lipids on porcine goblet cell homeostasis are varying. For instance, phosphatidylethanolamine supplementation elevates SAM pointed domain containing ETS transcription factor (Spdef) expression to promote goblet cell differentiation, thus improving intestinal barrier function in IUGR piglets (Wang et al., 2024a). By contrast, tributyrin reduces goblet cell number without affecting growth performance in weaned piglets (Tugnoli et al., 2020). In mice, phosphatidylcholine deficiency triggers necroptosis, a pro-inflammatory type of programmed cell death, of goblet cells, leading to intestinal injury and inflammation (Kennelly et al., 2021). The question of whether goblet cell necroptosis is present under inflammatory states, and its role in porcine intestinal inflammation are worth exploring to improve gut health in pigs.

In recent years, diverse plant extracts have been used to substitute antibiotics in swine production owing to their immunoregulatory and antioxidative properties (Gessner et al., 2017). Porcine goblet cell homeostasis can be improved by many plant extracts such as dihydroartemisinin, curcumin, and xylo-oligosaccharides (Niu et al., 2024; Xun et al., 2015). Similar effects are also achieved by supplementation with vitamins (Zhou et al., 2021), elements (Deng et al., 2021), bile acids (Song et al., 2021), acidifiers (Liu et al., 2024a), fibers (Pu et al., 2023), and multiple probiotics such as Lactobacillus rhamnosus GG and Bacillus amyloliquefaciens SC06 (Deng et al., 2022; Wang et al., 2024b) (Table 1). While the evidence for these benefits is substantial, the precise molecular mechanisms underlying most of these nutritional interventions require further investigation.

8. Potential roles of porcine goblet cells in nutrient digestion and absorption

In the small intestine, luminal nutrients are digested by various digestive enzymes and subsequently absorbed by enterocytes (Yu et al., 2024). Although goblet cells, as secretory epithelial cells, do not directly participate in the digestion and absorption of nutrients, they can affect these processes indirectly through multiple ways (Fig. 4). In piglets with postnatal growth retardation, the small intestinal goblet cell number is significantly lowered, and restoration of goblet cell homeostasis effectively improves piglet growth performance, counteracting growth retardation (Wang et al., 2024a), and suggesting the potential connection between goblet cells and nutrient utilization. Furthermore, goblet cells have been demonstrated to exert nutrient sensing capabilities as fructose treatment markedly enhances the expression of fructose-responsive genes glucose transporter 5 (Glut5), glucose-6-phosphatase (G6Pase), and ketohexokinase (Khk) in goblet cell-enriched organoids (Kishida et al., 2017).

Fig. 4.

Potential roles of porcine goblet cells in the digestion and absorption of nutrients. Goblet cell-derived mucins constitute mucus, which contributes to microbiota colonization and the uniform distribution of digestive enzymes. Digested nutrients and microbiota-derived vitamins or SCFAs are absorbed by enterocytes. Goblet cell derived TFF3 effectively maintains barrier integrity, thereby facilitating nutrient absorption. Additionally, goblet cells sample luminal nutrients to dendritic cells, which is vital to maintain intestinal immune tolerance to nutrients. SCFAs = short chain fatty acids; TFF3 = trefoil factor family 3; GAPs = goblet cell-associated antigen passages.

Goblet cell-derived MUCs are key components of the intestinal mucus layer which is a critical microenvironment for intestinal nutrient digestion and absorption. There are multiple digestive enzymes in mucus layer, such as lipase and trypsin, and the uniform distribution of digestive enzymes in mucus layer promotes the efficient digestion and absorption of luminal nutrients (Yu et al., 2024). Additionally, the mucus layer is essential for the maintenance of enterocyte homeostasis, thereby supporting efficient nutrient absorption. By covering the intestinal epithelium, the mucus layer physically separates luminal contents from underlying tissue, protecting the epithelium from injury and inflammation induced by microbiota and antigens. In mice, MUC2 deficiency severely compromises mucus layer integrity, enabling the direct contact between intestinal microbiota and the epithelium (Leon-Coria et al., 2021). Similarly, diarrheal piglets exhibit reduced goblet cell number, thinner mucus layers, and impaired intestinal barrier integrity, which all contribute to deficits in nutrient absorption and growth performance (Xia et al., 2022). Interestingly, several studies have demonstrated that increasing the mucus layer thickness does not affect the efficiency of nutrient absorption, indicating that nutrient diffusion in the mucus layer is not impaired by a thicker mucus layer (Morita et al., 2006, 2008).

It has been reported that goblet cell-derived TFF3 can maintain epithelial homeostasis via multiple effects, supporting nutrient absorption. Trefoil factor 3, in conjunction with MUCs, cooperatively protects the intestinal epithelium from various injuries caused by phytohemagglutinin, oleic acid, taurocholic acid, and Clostridium difficile toxin A (Kindon et al., 1995). Trefoil factor 3 facilitates the regeneration and migration of intestinal epithelial cells and exerts an anti-apoptosis effect on epithelium in mice (Belle et al., 2019; Dignass et al., 1994; Taupin et al., 2000). In pigs, TFF3 has been shown to significantly alleviate LPS-induced injury and inflammation in primary porcine intestinal epithelial cells (Chang et al., 2017), which could improve the nutrient absorption function of enterocytes. Notably, enterotoxigenic E. coli infection triggers robust TFF3 expression in piglets (Zhang et al., 2017), suggesting the protective role of goblet cells in nutrient absorption.

The mucus layer can be colonized by commensal microbiota which play important roles in nutrient degradation and synthesis (Rowland et al., 2018). In diarrheal piglets, the decrease in in the number of goblet cells or thickness of the mucus layer alters the composition and structure of commensal microbiota in the microbiome (Xia et al., 2022). Commensal microbiota utilize luminal nutrients and MUC O-glycan to produce beneficial metabolites such as SCFAs and vitamins, which not only provide energy for the intestinal epithelium but also help to maintain intestinal barrier integrity (Wu et al., 2021). Diarrheal piglets display an abnormal profile of MUC O-glycan with shorter O-glycan chains more prevalent when compared to healthy piglets, and fermentation of MUCs from diarrheal piglets yields fewer SCFAs such as acetate, propionate, and butyrate (Xia et al., 2022). Commensal microbiota are of great necessity for metabolizing primary bile acids to generate secondary bile acids, which contribute to the digestion and absorption of lipids, as well as intestinal barrier integrity through regulating bile acid receptors (Collins et al., 2023). In diarrheal piglets, intestinal microbiota disorders induced by goblet cell defects reduce the abundances of lithocholic acid and hyodeoxycholic acid (Xia et al., 2022). Notably, an abnormal bile acid profile in the porcine small intestine impairs lipid absorption as demonstrated by the elevation of free fatty acid concentration in colonic contents (Mao et al., 2025). Taken together, porcine goblet cells could also affect nutrient digestion and absorption through manipulation of commensal microbiota and their metabolites.

It has been reported that GAPs deliver innocuous luminal antigens, which include nutrients, to lamina propria dendritic cells, thereby maintaining intestinal tolerance to nutrients (McDole et al., 2012). Goblet cell-associated antigen passage-mediated intestinal tolerance prevents inflammation in response to luminal nutrients, which lays a foundation for nutrient digestion and absorption. In pigs, the presence of GAPs in the small intestine was proved (Fig. 2). The effect porcine GAPs have on nutrient digestion and absorption needs to be further investigated in future work.

9. Conclusions and perspectives

From a practical standpoint, porcine goblet cells are not merely passive barrier components but active regulators of gut health and productivity. Firstly, preserving goblet cell homeostasis emerges as a viable strategy to enhance disease resistance, particularly against common enteric pathogens like PEDV and ETEC. Secondly, the profound impact of goblet cells on the gut microenvironment positions them as critical mediators of nutrient utilization efficiency. Therefore, dietary formulations and management should consider their impact on goblet cell biology. In swine production, developing non-invasive biomarkers (e.g., MUC glycan profiles in feces) to monitor goblet cell function could facilitate proactive health management. Future studies should focus on validating this strategy and evaluating their cost-effectiveness.

Antigen presentation mediated by GAPs in goblet cells is of similar importance to intestinal and systemic protection. This paper demonstrated the presence of GAPs in the small intestine of pigs, laying a solid foundation for the further research on GAP-mediated intestinal health in 50-d-old growing pigs. Unscrambling the developmental changes of porcine GAPs, especially between the birth and post-weaning stages, is a task worth conducting to put forward potential strategies for improving gut health targeting GAPs. In swine production, porcine goblet cell homeostasis can be impaired by various negative factors such as weaning, infection, and mycotoxin. However, it has been shown diverse nutrients have a beneficial effect on porcine goblet cell abundance and MUC production and can mitigate negative effects. Hence, it is feasible to utilize appropriate nutrients to counter goblet cell disorders induced by negative factors in swine production.

CRediT authorship contribution statement

Chenbin Cui: Writing – original draft, Investigation. Jiaxi Tang: Investigation. Jing Hou: Investigation. Jianjian Zhang: Investigation. Min Tian: Investigation. Kaiguo Gao: Funding acquisition. Li Wang: Validation. Zongyong Jiang: Validation. Xuefen Yang: Supervision, Funding acquisition.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.