Epithelial-neuronal-immune cell interactions: implications for immunity, inflammation, and tissue homeostasis at mucosal sites

Abstract

The epithelial lining of the respiratory tract and intestine provides a critical physical barrier to protect host tissues against environmental insults including dietary antigens, allergens, chemicals, and microorganisms. In addition, specialized epithelial cells directly communicate with hematopoietic and neuronal cells. These epithelial-immune and epithelial-neuronal interactions control host immune responses and have important implications for inflammatory conditions associated with defects in the epithelial barrier, including asthma, allergy, and inflammatory bowel diseases (IBD). In this review, we discuss emerging research that identifies the mechanisms and impact of epithelial-immune and epithelial-neuronal crosstalk in regulating immunity, inflammation, and tissue homeostasis at mucosal barrier surfaces. Understanding the regulation and impact of these pathways could provide new therapeutic targets for inflammatory diseases at mucosal sites.

Introduction

Epithelial cells at mucosal sites act as a protective physical barrier against the external environment. In the airway and gastrointestinal tracts, epithelial cells selectively allow gaseous exchange and nutrient absorption, respectively, while they are in constant contact with diverse external environmental stimuli including dietary antigens, allergens, chemicals, and/or commensal and pathogenic microorganisms. Therefore, the functions of epithelial cells at these tissue sites are critical, and defects in epithelial barrier functions are associated with multiple infectious and inflammatory diseases including asthma (1–5), allergy (1,5–7), and inflammatory bowel diseases (IBD) (8–10). In addition to promoting barrier functions, epithelial cells are also important sensors of the environment, recognizing signals from commensal and pathogenic microbes, food, pollutants, and other particles (5,6,11,12). The ability of epithelial cells to sense, integrate, and respond to internal signals from the immune and nervous systems has also become appreciated (13–18). In this review we discuss emerging insights into how epithelial cells communicate with immune cells and the nervous system to regulate immunity, inflammation, and tissue homeostasis in the airway and intestine. Specifically, we provide an overview of the structure and functions of epithelial cells in the lung and intestine followed by a discussion on how epithelial cells regulate barrier immune responses. Lastly, we elaborate on recent findings regarding how immune cells and neurons regulate epithelial cell function at mucosal sites.

Structure and function of the epithelial lining

The epithelial lining of the respiratory and intestinal tracts is composed of a single layer of cells bound by tight junctions to form a physical barrier surface to the external environment (6,10,19). The respiratory tract is comprised of multiple airway passages including the nasal cavity, trachea, and lungs (20,21). In the lungs, alveoli comprise a layer of epithelial cells and an extracellular matrix surrounded by capillaries, forming the respiratory membrane for the exchange of gases (22). In the small intestine, epithelial cells form the crypt-villus axis and facilitate nutrient absorption, whereas the epithelium in the cecum and large intestine lacks villi and are critical in water absorption and other physiologic functions (23,24). Notably, epithelial cells at different regional tissue sites of either the lung or intestine express specific transcriptional gene signatures, supporting their specialized functions in different organs (25–29). Further, as discussed below, airway and gut epithelial cells in distinct tissue microenvironment are heterogeneous, exhibiting specialized functions.

Intestinal epithelial cell subsets

In the intestine, most epithelial cells can be broadly characterized as absorptive, having the capacity to absorb luminal contents, or secretory, releasing cytokines, chemokines, growth factors and other molecules specific to their functions. Pluripotent epithelial stem cells lie at the base of the crypts, giving rise to transit-amplifying cells that migrate up the crypt-villus axis and differentiate into distinct specialized cell types before eventually being exuded into the lumen (24). The majority of gut epithelial cells are enterocytes, which control water and nutrient absorption (6,10). Other specialized cell types include secretory cells such as Paneth cells and goblet cells. Paneth cells remain at the base of the crypts in the small intestine, surrounding the stem cell niche and secreting antimicrobial peptides (AMPs), while goblet cells produce mucins and other bioactive molecules, contributing to the luminal mucosal barrier that protects the epithelial cells from microbial penetration (30,31). Goblet cells can also produce AMPs and deliver antigen to dendritic cells (30,32–35). Other intestinal epithelial cell subsets include enteroendocrine cells (EECs), a heterogenous cell population that secrete hormones, neuropeptides, and neurotransmitters and that is known to interact with gut-innervating neurons (36–39). In contrast, chemosensory tuft cells, which recognize environmental cues via taste and other G-protein-coupled receptors, are important in sensing dietary contents and microbiota-derived products and can promote type 2 inflammation following helminth infection via their production of IL-25 and eicosanoids (40–44). Lastly, microfold (M) cells located on Peyer’s patches take up luminal antigens and deliver them to specialized dendritic cells (45–47).

Airway epithelial cell subsets

In the airway, epithelial cells also form a major barrier surface between the host and outside environment (3,48,49). The epithelial cells that line the alveoli are divided into two subtypes, type 1 pneumocytes and type 2 pneumocytes. Type 1 pneumocytes are flat in shape and thin, involved in gas exchange between the alveoli and the capillaries (22). Type 2 pneumocytes are cuboidal in shape and secrete pulmonary surfactant which reduce surface tension in the alveoli (22). Goblet cells are also present in the airway, where they secrete mucins to trap environmental molecules including dust particles and microbes that enter the respiratory tract (3,49). Ciliated epithelial cells are a dominant respiratory epithelial cell subset, controlling mucociliary clearance that is essential for expelling microbes and particles (21,48,50). Other airway epithelial cell subsets include stem-cell–like progenitor cells known as basal cells (51); pulmonary neuroendocrine cells (PNECs), innervated cells known to produce neuropeptides (52); ionocytes, a newly-identified cell type that highly express cystic fibrosis transmembrane conductance regulator and respond to osmotic stress (53); and chemosensory tuft cells, also known as brush cells or solitary chemosensory cells in the airway. Described in further detail below, airway tuft cells, which are found in the trachea and nasal airways but not the lungs at steady state, also express immune molecules such as IL-25 and eicosanoids (20).

The epithelial cell subsets present in the intestine and lung maintain barrier homeostasis by integrating signals from the external environment as well as signals from other cell types, including immune cells and neurons that underlie the epithelial layer. In both the intestine and the lungs, epithelial cells possess an array of pattern-recognition receptors (PRRs) including Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs) that can detect pathogen- or danger-associated molecular patterns (PAMPs/DAMPs) which has been reviewed elsewhere (6,54,55). In this review, we focus on new paradigms on epithelial regulation of immune cells, followed by immune and neuronal regulation of epithelial cells.

Epithelial regulation of immune responses

As described above, the airways and intestinal tract contain a variety of heterogenous epithelial cell types that can enact immune functions. This section will briefly discuss our current understanding of how non-specialized epithelial cells such as enterocytes and airway epithelial cells communicate with immune cells, followed by newly discovered mechanisms of how specialized epithelial cells including tuft cells and PNECs can directly modulate immune cells at mucosal barrier sites, shaping protective and pathologic responses.

Epithelial cell regulation of immunity and inflammation

Epithelial cells can secrete various cytokines to activate innate and adaptive immune cells in the context of modular type 1, type 2, and type 3 immune responses. Some common type 1/type 3 cytokines produced by epithelial cells during infection and tissue damage include Interleukin-1 (IL-1), IL-8, IL-18, tumor necrosis factor-α (TNFα), and transforming growth factor-β (TGFβ) (56–63). These cytokines play crucial roles in activating stromal cells and recruiting immune cells including neutrophils and monocytes, conferring protection against pathogens and contributing to tissue inflammation and repair (56–59,61,63)^,. During tissue damage elicited by allergens or helminth parasite infections, epithelial cells promote type 2 responses through production of chemokines, cytokines, and alarmins, including thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 (also produced by stromal cells (64–66)). These factors can recruit immune cells, such as group 2 innate lymphoid cells (ILC2s) and eosinophils, contributing to allergic inflammation or anti-helminth immunity and tissue repair (40–42,67–73).

Recent studies have shown that in addition to cytokines, vitamins such as retinoic acid (RA) produced from epithelial cells can also play critical role in immune cell homeostasis (74). For example, survival of intestinal eosinophils is directly enhanced by epithelial-derived RA (Figure 1A), an important immunomodulatory molecule known to regulate immune tolerance (74–76). Interestingly, this process is additionally regulated by the microbiota, as the bacterial species F. rodentium can suppress RA, contributing to the maintenance of normal eosinophil levels. However, whether F. rodentium controls epithelial RA production via metabolite release or directly interacting with epithelial cells remains unknown.

Figure 1.

Epithelial-immune cell interactions at gastrointestinal and respiratory barrier sites. (A) ILC3 production of IL-22 can enhance secretory epithelial lineages including goblet cells in vitro. Goblet cells in turn can further boost IL-22 from ILC3s, driven by goblet cell expression of Dll1 and Dll4 acting on the Notch1 receptor on T-bet⁺ NKp46⁺ ILC3s. In the small intestines, ILC2-derived IL-9 promotes Paneth cell metaplasia and the accumulation of secretory cells such as goblet cells and tuft cells. Epithelial-derived retinoic acid enhances the survival of eosinophils. Eosinophils, in turn, can negatively regulate epithelial proliferation and MHC class II expression. This process is in part mediated by eosinophils suppressing intraepithelial lymphocyte expression of IFN-γ, which promotes epithelial proliferation and MHC class II expression. Commensal bacteria such as F. rodentium also regulate this eosinophil-epithelial circuit by suppressing RA to negatively regulate eosinophil survival. (B) Pulmonary tuft cells expand in response to viral infection, although the function of this is unknown. Airway tuft cells also produce immunomodulatory factors in response to aeroallergens, leading to the expansion of goblet cells, type 2 cytokine-producing ILC2s, eosinophils, and CD103⁺ dendritic cells. Also in the airway, eosinophil extracellular traps promote epithelial cell production of the alarmins TSLP and IL-33 as well as PNEC production of CGRP, which activates ILC2 cytokine production, and GABA, which induces goblet cell hyperplasia during early postnatal OVA administration. Early life myeloid cell-derived IL-1β leads to increased expression of markers associated with basal cell differentiation.

Intestinal tuft cells in anti-helminth immunity and food allergy

In addition to the immunoregulatory functions of epithelial cells, specialized epithelial cells have distinct mechanisms of controlling immune cells. Found at mucosal sites, including the gastrointestinal and airway tracts, and identified by their long, blunt microvilli, tuft cells express taste receptor machinery (77,78), but their functions were poorly characterized until recently. Seminal studies in 2016 identified that intestinal tuft cells produce IL-25, which activates ILC2 function, resulting in goblet cell hyperplasia that can contribute to expulsion of helminth parasites (40–42). While the function of tuft cells in anti-helminth immunity is now established, recent studies also identified a role of tuft cell-secreted IL-25 in allergic reactions. In a model of tape stripping-induced food allergy in mice where systemic IL-33 released from injured skin promotes allergic responses in the intestine (79), it was recently shown that tuft cell-derived IL-25 synergized with keratinocyte-derived IL-33 to expand and activate intestinal ILC2s (80). In this model, tape stripping of the skin led to an increase in small intestinal tuft cells and epithelial Il25 expression, of which tuft cells are the dominant source (41). Furthermore, conditional knockout of intestinal epithelial IL-25 or ILC2-expressed IL-25 receptor (Il17rb) abolished the tape stripping-induced increase in small intestinal ILC2s and ILC2-derived Il4 and Il13 expression (80). ILC2-derived IL-4/13 signaling contributes to a feed-forward circuit to further promote intestinal tuft cells (41), and furthermore, ILC2-derived IL-4 promoted mast cell responses in the intestine, leading to food anaphylaxis responses (80). Together, these studies show that intestinal tuft cells can be primed by distal injury or inflammation, such as skin barrier damage, contributing to food allergy pathogenesis in the intestine.

Functions of airway tuft cells during asthma and viral infection

Other recent advances in how tuft cells can alter immune responses have focused on airway tissues. In the healthy airway, tuft cells can be found in the nasopharynx tissues and trachea, but are rare or absent in the lungs (43,81,82). Similar to the intestine, nasal and tracheal tuft cells can produce cysteinyl leukotrienes (cysLTs), IL-25, and prostaglandin E2 in response to airway inflammation (83–85) (Figure 1B). Activation of these airway tuft cells via inhalation of the aeroallergen Alternaria was found to lead to the accumulation of CD103⁺ dendritic cells, type 2 cytokine-producing ILC2s, eosinophils and goblet cells in the lung (84). These responses were abrogated in mice deficient in tuft cells or with a tuft cell-specific depletion of Ltc4s, the enzyme responsible for LTC₄ production, indicating that tuft cells can mediate type 2 immune responses via lipid mediator signaling (84).

While tuft cells are rare in the lung at steady state, the accumulation of tuft cells has been identified following viral infections, including H1N1 (81), influenza A (IAV) (82,86), and rhinovirus (RV) (87) (Figure 1B), as well as after bleomycin-induced injury (86). In the context of IAV, tuft cell expansion occurred independently of IL-25 and IL-4/13 signaling (86), suggesting an alternative pathway to tuft cell differentiation. Whether airway tuft cells have any direct role in antiviral immunity during pulmonary viral infection remains unclear, as the absence of tuft cells did not affect disease parameters including weight loss, goblet cell differentiation, or Krt5⁺ epithelial remodeling (86,88). However, tuft cell responses during acute viral infections can have implications for development of allergy. For example, a recent study has shown that during RV infection, tuft cell deficiency led to reduced type 2 cytokine expression and mucus metaplasia, which later blocked the development of type 2 inflammatory responses in mice post-infection (87), building on previous work showing that tuft cells are involved in allergic airway diseases following Alternaria exposure (83,84). However, more work is needed to establish the functions, if any, of tuft cells during acute and chronic viral infections.

PNEC and goblet cell regulation of ILCs

PNECs have also been implicated in the pathogenesis of airway disease (52,89). Lung tissues from human asthma patients exhibit increased densities of PNECs, particularly calcitonin gene related peptide-expressing (CGRP⁺) PNECs (89). Furthermore, mice deficient in PNECs exhibit reduced type 2 inflammatory responses in a model of early postnatal ovalbumin (OVA) administration (89). This was shown to be mediated by PNEC production of both CGRP, which promoted ILC2 cytokine production, and γ-aminobutyric acid (GABA), which induced goblet cell hyperplasia (89) (Figure 1B). Together, these data suggest that PNECs are important regulators of type 2 inflammation in the airways.

A recent study also implicates intestinal goblet cells in the regulation of group 3 innate lymphoid cell (ILC3) responses (90). Intestinal organoids enriched in goblet cells promoted IL-22 production by ILC3s compared to baseline organoids or organoids enriched in Paneth cells. This increased production of IL-22 by ILC3s was shown to be driven by goblet cell expression of the Notch ligands Delta-Like-Canonical-Notch-Ligand (Dll) 1 and Dll4 acting on Notch1 receptor on ILC3s (90) (Figure 1A). Considering that the dysregulation of ILC3 responses is associated with IBD (91), this pathway could act as a potential therapeutic target for management of intestinal inflammation.

To summarize, epithelial regulation of immune cells plays an important role in immune cell regulation, inflammation, and tissue repair in the airway and intestine.

Immune regulation of epithelial cell responses

While epithelial cells can regulate diverse immune cell types (1–4,6,10,12,19,92–95), several innate immune cells have been shown to directly regulate the activation or regeneration of the epithelial cells. Here we discuss recent findings on how innate immune cells regulate epithelial cell development and function.

Regulation of epithelial cells by ILCs

ILCs are lymphocytes that lack antigen-specific receptors and act as important regulators of immunity, inflammation, tissue homeostasis, repair, and regeneration (96,97). The ILC family is composed of T-bet⁺ group 1 ILCs (ILC1s), natural killer (NK) cells, GATA3⁺ ILC2s, and RORγt⁺ ILC3s (96–99). ILC2s are associated with type 2 immune responses against exposure to helminths and allergens, producing type 2 cytokines such as IL-4, IL-5, and IL-13 to drive intestinal epithelial cell proliferation, goblet and tuft cell differentiation, and mucus secretion at mucosal sites (40–42,96,98,100,101). Alarmin-activated ILC2s also produce amphiregulin, which promotes colonic mucus production and epithelial integrity after helminth infection, dextran sodium sulfate (DSS)-induced colitis, or influenza virus infection (102–104). Further, following allergens or helminth infection, tuft cell-derived IL-25 promotes ILC2 responses (40–42). Recently, it has also been shown that ILC2s can also modulate Paneth cell responses (105). In a murine model of chronic myelogenous leukemia (106), ILC2s produced IL-9 that promoted the Paneth cell metaplasia and the accumulation of secretory cells including goblet cells and tuft cells in the small intestine (105). This pathway also led to changes in epithelial cell function, including increased production of IL-33 (105). While Paneth cell metaplasia has been shown in human diseases including IBD (107) and colorectal cancer (108), whether this ILC2–IL-9–epithelial cell circuit is clinically relevant in these conditions remains to be determined.

ILC3 regulation of epithelial cells also plays a critical role in regulating intestinal damage, inflammation, and tissue repair. For example, ILC3-derived IL-22 has previously been shown to increase the antimicrobial and tissue-protective functions of the intestinal epithelium via the activation of STAT3 to promote intestinal stem cell regeneration, the mucus barrier, and AMP production (109–116). While ILC3-derived IL-22 can promote intestinal stem cell regeneration (111,112), a recent study also found that this signaling pathway can also promote differentiation of secretory epithelial cell lineages (90). In a small intestinal epithelial organoid co-culture system, ILC3s were shown to promote the differentiation of intestinal epithelial secretory cells, amplifying gene expression associated with goblet and Paneth cells compared to organoids without ILC3s (90) (Figure 1A). Notably, translational studies identified a link between dysregulated secretory epithelial cell responses and inflammatory diseases such as IBD, including a reduction in the proportion of secretory cells such as goblet cells or their progenitors (117,118). While further studies are needed to confirm the mechanisms that regulate these responses in vivo, these findings highlight a signaling axis between ILC3s and intestinal epithelial cells that could have implications in IBD and other chronic inflammatory diseases at mucosal barrier sites.

Myeloid cell regulation of pulmonary epithelial cell development

In addition to ILCs, myeloid cells can also regulate epithelial cell development and responses. After injury, monocytes and macrophages secrete mediators such as TGF-β and IL-10 to suppress inflammation and enhance the wound repair response (119,120), while during helminth infection, IL-4- and IL-13-activated macrophages are important for the resolution of inflammation and tissue repair (119–121). In the steady state adult lung, alveolar macrophages and epithelial cells regulate each other to inhibit the development of unwanted inflammation (94). Recently, gene expression profiling of immune cells in fetal human lungs using single cell RNA sequencing and immunohistochemistry revealed an enrichment of myeloid cells in direct contact with or in close proximity to SOX9⁺ epithelial progenitor cells in the distal epithelial tips, which can differentiate into all other airway lineages (122,123). Ex vivo culture of dendritic cells and macrophages isolated from human fetal lungs found that these cells produced a variety of cytokines, including IL-1β, and in vitro stimulation of epithelial organoids derived from the fetal lung tissue with IL-1β for 14 days led to increased expression of markers associated with airway epithelial differentiation, specifically that of basal cells but not secretory cells (122). Overall, while not directly shown, this study suggests that myeloid-specific IL-1β can control fetal lung epithelial cell differentiation in humans (Figure 1B). Notably, this study also highlighted that IL-13 stimulation led to a distinct effect on epithelial cell differentiation, which could have implications for IL-13–producing immune cells such as ILC2s in airway development.

Eosinophil regulation of epithelial cells

Intestinal eosinophil responses are typically associated type 2 inflammation following exposure to allergens or helminth infections (121,124,125). In addition to their production of IL-5 and other bioactive molecules that can promote type 2 inflammation (121,124,125), eosinophils have recently been found to negatively regulate intestinal epithelial cell turnover and MHC class II expression, thus regulating inflammatory and tissue injury responses (Figure 1A) (74). Eosinophil-deficient PHIL mice exhibited increased intestinal epithelial turnover and MHC class II expression compared to wild type mice, suggesting that eosinophils negatively regulate epithelial cell proliferation and activation (74). Further, in an anti-CD3 antibody-mediated model of inflammation and epithelial damage, PHIL mice developed worse intestinal damage compared to wild type mice, indicating that the lack of eosinophil regulation of epithelial cells leads to improper recovery from injury (74). This pathway was also shown to be mediated by eosinophil-mediated suppression of intraepithelial lymphocyte production of IFN-γ, which is known to induce intestinal epithelial cell proliferation (126) and MHC class II expression (127). Whether eosinophils regulate epithelial cell proliferation or MHC II expression during type 2 inflammation, such as during helminth infection or allergen exposure, remains to be investigated.

Eosinophils have also been shown to regulate epithelial cell function in the lung. Eosinophil extracellular traps and eosinophil peroxidase were found to promote epithelial production of alarmins such as IL-33 and TSLP, as well as activate PNEC production of CGRP and GABA to amplify allergic immune responses, including goblet cell hyperplasia, enhanced mucus production, and increased of type 2 cytokines from the bronchoalveolar lavage fluid (BALF) (128,129) (Figure 1B). Considering that eosinophil extracellular traps from the BALF are associated with the severity of asthma in human patients (128,129), this mechanism indicates a possible pulmonary immuno-epithelial circuit in the context of type 2 inflammation in the airways. Overall, these studies identify innate and adaptive immune cells as important for regulators of the epithelial cell response to infection, injury, and environmental insults.

Neuronal regulation of epithelial cell function

Epithelial cell activation can be mediated by signals beyond the immune system including other neighboring cell types such as neurons. Extrinsic and intrinsic nerves innervate the intestinal epithelium (130), with EECs (36,37,131) and tuft cells (132–134) known to have direct interactions with and/or be in close proximity to nerves. The respiratory tract is also innervated by afferent and efferent nerves that travel along the vagus nerve (135,136). As in the GI tract, airway tuft cells (137,138) and PNECs (13) are also known to contact nerve fibers directly. Previous studies reported that neurons can support intestinal epithelial cell responses, including enhancing the expression of genes associated with intestinal stem and transit-amplifying cells, while reducing the expression of genes associated with absorptive, goblet, and Paneth cells in vitro (139). Neuronal signals can also support intestinal tuft cell survival in vitro and in vivo (140). As outlined below, recent work has revealed that neuronal signals can regulate specific epithelial cell subsets, including goblet cells, tuft cells, EECs, and PNECs as well as promote the regeneration of epithelial cells.

Neuronal regulation of goblet and secretory cell-associated antigen passages

Intestinal secretory cells such as goblet cells have been shown to form goblet and secretory cell-associated antigen passages (GAPs/SAPs), wherein epithelial cells can deliver luminal antigens to lamina propria immune cells (32,33,141). GAP/SAP formation can contribute to the maintenance of tolerance to dietary antigen (32,33,142) as well as promote IgE-mediated food-induced anaphylaxis (141). In addition, the neurotransmitter acetylcholine (ACh) plays an important role in the formation of GAPs at steady state (142). Recent studies revealed that Ach-mAChR4 signaling in the small intestine and ACh-mAChR3 signaling in the distal colon specifically regulate the steady state formation of GAPs, but not mucus secretion, another major function of goblet cells that can be induced by ACh signaling (34,143) (Table I, Figure 2A). Notably, supraphysiologic ACh signaling using carbamylcholine (CCh), an ACh analogue, induced both GAP formation and mucus secretion, both mediated by mAChR1 in the small and large intestine (34). Together, these studies identify one mechanism in which the neurotransmitter ACh can regulate luminal antigen delivery by epithelial cells to immune cells.

Table I:

Neuronal regulators of mucosal epithelial cells

Signal	Tissue	Signal target	Effect	Reference
ACh	Mouse SI	Goblet cell mAChR4, mAChR1	Promotes steady state GAP formation	34,142
	Mouse distal colon	Goblet cell mAChR3, mAChR1	Promotes steady state GAP formation	34
	Mouse SI	Goblet cell mAChR1, mAChR3	Mucus secretion	34,143
	Mouse colon	Goblet cell mAChR1	Mucus secretion	34
	Drosophila midgut	Epithelial cell nAChRβ3	Regulates Ca2+ levels following DSS-induced injury to promote epithelial maturation and ion balance, facilitating intestinal epithelial recovery	160
	Drosophila midgut	Enterocyte nAChR	Promotes epithelial barrier function	162
CGRP	Mouse SI	M cells	Limits the density of M cells in Peyer’s patch FAE during Salmonella infection, controlling susceptibility to Salmonella	150
	Mouse colon	Goblet cell RAMP1/CALCRL	Promotes colonic mucus production, protecting against DSS-induced colitis pathology	158
IL-18	Mouse colon	Unknown	Protects against bacterial infection by driving goblet cell-derived AMP expression	151
NT4	Mouse lung	PNECs	Promotes PNEC innervation at steady state and after allergen exposure and, with GABA, promotes mucus production after OVA challenge	159
GABA	Mouse lung	Goblet cells	Induces goblet cell hyperplasia	89, 159
Norepinephrine	Mouse SI	ILC3 Adrb1/2	Promotes ILC3 production of IL-22 to indirectly enhance epithelial proliferation	163
	Mouse SI	Epithelial cell Adra2a	In vitro, promotes mouse intestinal organoid growth and epithelial stem cell gene expression; in vivo, increases SI crypt depth and villus height and facilitates anastomotic wound healing	164

Figure 2.

Epithelial-neuronal interactions at gastrointestinal and respiratory barrier sites. (A) While ACh signaling via epithelial mAChR4 in the small intestines or mAChR3 in the large intestines regulates the formation of GAPs but not mucus secretion, ACh signaling via epithelial mAChR1 promotes both GAP formation and mucus secretion. Nociceptor-derived CGRP acts directly on goblet cells to increase mucus release in the colon, protecting mice from DSS-inducted damage. During DSS-induced intestinal damage, ACh from ARCENs signal on intestinal epithelial cells via nAChRβ3 to promote epithelial maturation and repair. Norepinephrine from adrenergic nerves promotes small intestinal epithelial recovery after IR-induced injury by inducing IL-22 from ILC3s. Adrenergic signaling directly on epithelial cells also boosts epithelial proliferation. (B) On Peyer’s patches, nociceptor-derived CGRP inhibits the expansion of M cells in the ileum, protecting mice from STm infection. (C) In the lungs, PNECs regulate local innervation and goblet cell mucus secretion via NT4 and GABA.

Although the sources of ACh inducing intestinal GAP formation have not been determined, it was reported that sensory neurons mediate GAP formation in the conjunctival epithelial layer in response to pollen and soluble antigens (144). While topical application of OVA with ragweed pollen shells to the conjunctiva elicited eosinophilic conjunctivitis, topical lidocaine to inhibit axonal electrical transmission significantly diminished GAP formation and antigen uptake (144). Unlike intestinal GAP formation, however, mAChR signaling was not essential for conjunctival GAP formation, as blocking mAChR signaling using atropine, a pan-mAChR antagonist, did not significantly alter the number of GAPs, and CCh only marginally increased the number of GAPs (144). Instead, ablation of trigeminal nerves using electrocoagulation almost completely abrogated GAP formation, suggesting that another sensory neuron-derived factor may induce GAP formation in the conjunctiva.

Neuronal regulation of epithelial cell defense against pathogens

CGRP, a nociceptive neuropeptide, is another neuronal signal that has been shown to regulate immune responses (145–149) and directly act on epithelial cells to influence immunity against enteric infections. For example, small intestinal pain-sensing neurons, nociceptors, release CGRP in response to bacterial infection of Salmonella enterica serovar Typhimurium (STm) to limit the density of M cells in Peyer’s patch follicle-associated epithelium (FAE) which protects against infections (150) (Table I, Figure 2B). Ablation of Nav1.8⁺ or TRPV1⁺ nociceptors led to an increase in the frequency of GP2⁺ M cells, which are required for Salmonella infection, resulting in an increase in STm burden (150). TRPV1⁺ neurons also were shown to directly respond to STm by releasing CGRP (150). Further, deletion of the CGRPα isoform of CGRP in Calca^−/− mice exhibited increased levels of ileal M cells and STm burden, suggesting that nociceptor-derived CGRPα regulates M cell frequency to control susceptibility to STm, although the mechanism of how CGRP impacts M cells remains unclear. Of note, IL-18 has also been shown to be an enteric nervous system-derived protein that indirectly leads to epithelial changes to protect against STm infection by promoting goblet cell expression of AMPs (151) (Table I). Considering that intestinal neurons can also produce other neuropeptides such as neuromedin U (NMU) and vasoactive intestinal peptide (VIP), which promote ILC2 type 2 cytokine production (152–157) that can control epithelial cell proliferation and differentiation (40–42,96,98,100,101), there are several pathways by which intestinal-innervating nerves can drive epithelial changes to promote immunity.

Neuronal regulation of epithelial cell function in inflammation

Beyond mediating defense against bacterial pathogens, nociceptor-derived CGRP can directly regulate goblet cell responses, which express the CGRP receptor comprised of the components RAMP1 and CALCRL (158). Nav1.8⁺ nociceptor ablation, Calca deficiency, or intestinal epithelial cell-specific Ramp1 deficiency led to reduced colonic mucus thickness in mice (158) (Table I, Figure 2A). Alternatively, chemogenetic activation of nociceptors or administration of exogenous CGRP led to an increase in mucus layer thickness (158). Further, while nociceptor ablation or epithelial cell-specific Ramp1 deficiency conferred susceptibility to DSS-induced colitis, CGRP administration rescued nociceptor-ablated mice from exacerbated DSS-induced intestinal pathology, suggesting the nociceptor signaling to epithelial cells is necessary for host protection against gut inflammation (158).

In the lung, PNECs can control mucus production in the context of early life allergen exposure (159). Mice deficient in the PNEC-derived growth factor neurotrophin 4 (NT4) did not exhibit an OVA-induced increase in mucus production despite an otherwise normal immune response to neonatal OVA sensitization and challenge (Table I, Figure 2C). NT4-deficient mice also had reduced levels of innervated PNECs at steady state and after allergen exposure (159). After culturing various NT4^−/− lung samples with OVA plus another neurotransmitter, only stimulation with GABA, previously shown to induce goblet cell hyperplasia (89), rescued Muc5ac production (159). In addition, PNEC-specific deletion of Gad1, an enzyme involved in GABA production, or Slc32a1, a GABA vesicular transporter, in mice resulted in reduced OVA-induced mucus production, similar to outcomes reported in NT4-deficient mice (159). Overall, this paper showed that NT4 and GABA from PNECs are necessary for mucus overproduction and PNEC hyperinnervation after early life allergen exposure. While the authors argue that the PNEC release of GABA is controlled by the neurons, further investigation is warranted to confirm this mechanism.

Direct neuronal regulation of intestinal epithelial cell recovery and repair

A number of studies recently established different mechanisms through which neuronal signals can support epithelial cell regeneration after injury. Using the Drosophila midgut as a model, a pathway in which cholinergic neurons control epithelial recovery after colitis-like injury was identified (160) (Table I, Figure 2A). Following DSS-induced intestinal damage in Drosophila, epithelial cells downregulated the expression of acetylcholinesterase, an enzyme that breaks down the neurotransmitter ACh, and upregulated the expression of nicotinic Acetylcholine Receptor β3 (nAChRβ3), an ACh receptor. Furthermore, Ca²⁺ levels that are known to be regulated by ACh signaling (161) were increased in ex vivo epithelial cells following injury or ACh administration compared to during homeostasis in an nAChRβ3-dependent manner (160). This signaling axis promoted epithelial maturation and ion balance, contributing to intestinal epithelium recovery. Depleting choline acetyltransferase (ChAT, the enzyme responsible for ACh production) from Drosophila enteric neurons, but not epithelial or immune cell lineages, led to increased proliferation of epithelial cells during recovery from injury and increased inflammatory cytokine expression (160). Related to these findings, ACh could also promote epithelial barrier function via enterocyte nAChR signaling in Drosophila (162) (Table I). Together, these data suggest that intestinal neurons, specifically termed anti-inflammatory recovery-regulating cholinergic enteric neurons (ARCENs), control epithelial repair via a bioelectric pathway mediated by Ca²⁺ and ACh signaling.

Other neuronal signals can also support epithelial barrier repair. Following irradiation (IR)-induced injury in mice, the density of gut-innervating adrenergic nerves in the small intestine was increased and promoted ILC3 production of IL-22 via norepinephrine signaling to indirectly enhance epithelial recovery (163) (Table I, Figure 2A). Denervating these adrenergic nerves led to reduced ILC3-derived IL-22, mediated by Adrb1/2 signaling, resulting in impaired epithelial proliferation (163). While this work did not address whether adrenergic nerves can directly affect intestinal epithelial cells, another study reported that the adrenergic receptor isoform α_2A (Adra2a) is broadly expressed in mouse and human intestinal epithelial cells, suggesting that this pathway could occur (Table I, Figure 2A) (164). Indeed, organoids stimulated with norepinephrine or the α_2-AR agonist UK 14,304 grew larger in size and exhibited increased expression of genes associated with intestinal stem cells and decreased expression of genes associated with differentiated cell types (164). In vivo gavage of the α_2-AR agonist UK 14,304 also led to increased small intestinal crypt depth and villus height, further suggesting that α_2-AR signaling controls stem cell function (164). In addition, in a model of anastomotic wound healing in mice, sympathetic denervation led to worse epithelial recovery from injury (164). However, further studies are required to confirm the direct epithelial-specific effects of α_2-AR signaling.

Altogether, these studies demonstrate that neurons innervate or are in close proximity to mucosal epithelial cells, and the crosstalk between these cells can control host homeostasis.

Concluding remarks

As the physical barrier between the outside world and the cells within the body, epithelial cells in the intestine and lung play an active role in sensing signals from both the external environment and adjacent internal cells and tissues to control physiological responses at steady state and following exposure to infection, injury, and repair processes. Different epithelial cell subsets can respond to these signals by releasing various effector molecules, engaging in a crosstalk with host immune cells and neurons. Further, while epithelial-immune-neuron interactions are pivotal in touch and pain sensation in the skin (165–168), recent studies described in this review have begun to illuminate the significance of this signaling axis at mucosal sites. Given the importance of proper epithelial barrier maintenance for diseases such as asthma (1–5), allergy (1,5–7), and IBD (8–10) and the ability of immune cells and neurons to regulate barrier integrity, the crosstalk of epithelial cells with immune cells and neurons likely plays a major clinical role in the context of inflammatory diseases. Furthermore, the ability of immune cells and neurons to regulate the function of specific epithelial cell subsets such as goblet cell mucus release or tuft cell prostaglandin and leukotriene production could have implications for type 2 inflammation-mediated diseases. While further studies are required to identify the different functions of neuronal signaling on mucosal epithelial cells during infection, injury, and inflammation, these findings are promising avenues for future therapeutic intervention strategies.

Advances in organoid technology will help further our understanding of airway and intestinal epithelial crosstalk with other cell types, as various groups develop organoid co-culture systems with immune cells (122,169–172) and neurons (173,174) to complement in vivo studies. These 3D in vitro systems will also be useful for screening potential therapeutic targets for translational research in inflammatory diseases associated with disrupted mucosal barrier function, such as IBD, allergies, and asthma, as discussed earlier. In addition, elucidation and functional characterization of how epithelial-immune and epithelial-neuronal interactions are regulated in different organs, within the same organ, and between organ systems could offer valuable new insights.

Given that mucosal epithelial cells can express a variety of receptors recognizing different microbiota-derived signals (6,12,21,23,28,44,48,54), and microbial-derived metabolites themselves are known to influence immune responses (175–178), future studies are needed to determine how the microbiota and/or microbiota-derived products influence the crosstalk between epithelial cells with neurons or immune cells, which could aid in the development of therapeutic strategies against acute and chronic inflammatory diseases in the gut and lung. Deepening our knowledge of the fundamental processes of how mucosal epithelial cells regulate and are regulated by other immune cells and neurons will be essential for informing translational research in this field.

Abbreviations used:

IBD

inflammatory bowel disease

AMPs

antimicrobial peptides

EEC

enteroendocrine cell

PNEC

pulmonary neuroendocrine cell

PRR

pattern-recognition receptor

TLR

Toll-like receptor

NLR

NOD-like receptor

RLR

RIG-I-like receptor

PAMP

pathogen-associated molecular patterns

DAMP

danger-associated molecular patterns

IL

interleukin

TNF-α

tumor necrosis factor-α

TGFβ

transforming growth factor-β

TSLP

thymic stromal lymphopoietin

ILC

innate lymphoid cell

ILC2

group 2 innate lymphoid cell

RA

retinoic acid

cysLTs

cysteinyl leukotrienes

IAV

influenza A

RV

rhinovirus

CGRP

calcitonin gene related peptide

OVA

ovalbumin

GABA

γ-aminobutyric acid

ILC3

group 3 innate lymphoid cell

Dll

Delta-Like-Canonical-Notch-Ligand

ILC1

group 1 innate lymphoid cell

NK

natural killer

DSS

dextran sodium sulfate

BALF

bronchoalveolar lavage fluid

GAP

goblet cell-associated antigen passage

SAP

secretory cell-associated antigen passages

ACh

acetylcholine

FAE

follicle-associated epithelium

NMU

neuromedin U

VIP

vasoactive intestinal peptide

NT4

neurotrophin 4

ARCEN

anti-inflammatory recovery-regulating cholinergic enteric neuron