Research progress on the relationship between Paneth cells-susceptibility genes, intestinal microecology and inflammatory bowel disease

Abstract

Inflammatory bowel disease (IBD) is a disorder of the immune system and intestinal microecosystem caused by environmental factors in genetically susceptible people. Paneth cells (PCs) play a central role in IBD pathogenesis, especially in Crohn's disease development, and their morphology, number and function are regulated by susceptibility genes. In the intestine, PCs participate in the formation of the stem cell microenvironment by secreting antibacterial particles and play a role in helping maintain the intestinal microecology and intestinal mucosal homeostasis. Moreover, PC proliferation and maturation depend on symbiotic flora in the intestine. This paper describes the interactions among susceptibility genes, PCs and intestinal microecology and their effects on IBD occurrence and development.

Key Words: Susceptibility gene, Paneth cells, Intestinal microecology, Inflammatory bowel disease

Core Tip: Inflammatory bowel disease (IBD) is a disorder of the immune system and intestinal microecosystem caused by environmental factors in genetically susceptible people. Paneth cells (PCs) play a central role in IBD pathogenesis, especially in Crohn's disease development, and their morphology, number and function are regulated by susceptibility genes. In the intestine, PCs participate in the formation of the stem cell microenvironment by secreting antibacterial particles and play a role in helping maintain the intestinal microecology and intestinal mucosal homeostasis. Moreover, PC proliferation and maturation depend on symbiotic flora in the intestine. This paper describes the interactions among susceptibility genes, PCs and intestinal microecology and their effects on IBD occurrence and development.

INTRODUCTION

Inflammatory bowel disease (IBD) is a disorder of the immune system and intestinal microecosystem caused by environmental factors in genetically susceptible people. Paneth cells (PCs) play a central role in IBD pathogenesis, especially in Crohn's disease (CD) development, and their morphology, number and function are regulated by susceptibility genes. In the intestine, PCs participate in the formation of the stem cell microenvironment by secreting antibacterial particles and play a role in helping maintain the intestinal microecology and intestinal mucosal homeostasis. Moreover, PC proliferation and maturation depend on symbiotic flora in the intestine. This paper describes the interactions among susceptibility genes, PCs and intestinal microecology and their effects on IBD occurrence and development.

IBD is a group of chronic, nonspecific inflammatory diseases that include CD and ulcerative colitis (UC). PCs, derived from intestinal pluripotent stem cells, are gradually growing columnar epithelial cells located at the junction of villi and crypts[1]. During differentiation and maturation, PCs migrate to the base of crypts and spread throughout the small intestine. PCs are pyramidal, and the cytoplasm at the “top” of the pyramid is full of coarse and large eosinophilic secretory granules. The main components are α-defensin, lysozyme, phospholipase A2 and other antibacterial substances that can be released into the intestinal cavity and play an important role in the natural defense of the small intestinal mucosa[2]. Due to the high secretion capacity of PCs and their close relationship with intestinal microecology, PC abnormalities and flora disorders often occur in the intestinal inflammatory response[3]. In recent years, PCs have been found throughout the gastrointestinal tract, including the stomach and colon. However, the distribution of PCs throughout the gastrointestinal tract occurs mainly as a response to mucosal inflammation, and PC presence in abnormal areas is called metaplasia. Colorectal PCs are widely found in UC and inflammatory enteritis[4].

In the past 20 years, the results of whole-genome scanning have revealed that IBD susceptibility genes are distributed on chromosomes 1, 3, 4, 5, 6, 7, 10, 12, 14, 16, 19 and X, among which 9 susceptibility genes associated with IBD were named IBD1-9: IBD1 on chromosome 16q, IBD2 on 12p13.2-q24.1, IBD3 on 6p, IBD4 on 14q11-q12, IBD5 on 5q31, IBD6 on 19p13, IBD7 on 1p36, IBD8 on 16p and IBD9 on 3p26[5]. Studies have shown that more than 200 gene loci are associated with IBD susceptibility, including more than 150 that increase the risk of CD[6]. Studies have shown that IBD susceptibility genes can affect the important physiological processes of PC, leading to abnormal PCs and promoting the occurrence and development of intestinal mucosal inflammation[7].

Changes in the intestinal microecology are involved in IBD pathogenesis, which mainly manifests as a flora imbalance, including flora diversity, species and abundance changes. Studies have shown that the abundance of Firmicutes and Bacteroidetes, which dominate the intestinal flora of IBD patients, decreases, while the proportion of Proteobacteria and actinomycetes increases[8]. Therefore, based on the above knowledge, this paper reviews the relationship among PCs, susceptibility genes, intestinal microecology and IBD.

THE ORIGIN AND METAPLASIA OF PCS

In 1745, German anatomist Johann Nathanael Lieberkuhn first described intestinal glands, or crypts, present in the intestines. In 1872, Gustav Schwalbe observed PCs in the crypt of the small intestine. In 1888, the Austrian physician Joseph Paneth described PCs graphically as a group of specialized cylindrical cells in the crypts of the small intestine epithelium, whose cytoplasm is filled with granular matter. The cells have been named PCs in honor of Dr. Paneth. PCs are rare cells in the small intestine that provide the host with protection against microbial invasion. Their function is the secretion of antibacterial proteins.

When bacteria or bacterial antigens invade the body, PCs secrete antibacterial molecules such as defensins between the villi of the bowel to help maintain the gastrointestinal barrier[9]. PCs are characteristic cells of the small intestinal gland, located at the bottom of the gland, the cells are pyramidal, and the top cytoplasm is full of coarse and large eosinophilic secretory particles. Under the electron microscope, the cytoplasm contains a large number of rough endoplasmic reticulums and developed Golgi complexes, and the secretory particles contain defensin and lysozyme, which can kill intestinal microorganisms. Most of the substances secreted by PCs are antibacterial proteins, which are expelled from the recess of the small intestine and dispersed into the mucosal layer to assist the mucosal immune barrier in its function. Later, PCs were found in the gastrointestinal tract, including the stomach, small intestineand colon[10]. However, PCs in the gastrointestinal tract are mainly found in response to mucous membrane inflammation, which is called metaplasia in abnormal colorectal regions. Paneth cell metaplasia is widespread in inflammatory enteritis.

PHYSIOLOGICAL FUNCTION OF PCS

Providing a niche for small intestinal stem cells in intestinal crypts

In adult mammalian tissues, the small intestine epithelium has a remarkable capacity for self-renewal. The renewal of the small intestinal epithelium depends on stem cells in the small intestinal crypt. Progenitor cells differentiated from small intestinal stem cells migrate from the bottom of the crypt to the small intestinal villi and continue to differentiate into goblet cells, plexus cells, neuroendocrine cells, intestinal epithelial cells and other cells[11]. After differentiation, these cells migrate from the crypt to the apex of the villi, where they gradually die and are replaced by new cells that migrate from the lower end. Intestinal epithelial cells have a life cycle of only four to five days, and this rapid self-renewal is thought to be essential for intestinal integrity. PCs are also derived from small intestine stem cells, but unlike other cells, PCs do not migrate upward after they are produced[12]. They always reside in the crypt, and the lifespan of this group of cells is more than 1 mo.

Intestinal stem cells are located at the base of the crypt, and recent research suggests that there may be two types of stem cells present. One type is crypt base columnar cells (CBC cells), which are spaced apart from PCs at the base of the crypt[13]. The target gene of Wnt, Lgr5, is the most representative marker of CBC cells and is expressed on the surface of CBC cells. The other type is static +4 cells, which are located above PCs, and the main markers include Bmi-1, Hopx, mTert and Lrig1. Little is known about +4 cells, but studies have shown that there is a close relationship between CBC cells and PCs[14].

The close spatial relationship between PCs and CBC cells has prompted speculation that PCs provide an important niche for stem cells[15]. However, one laboratory refuted this hypothesis. They created a transgenic mouse model in which PCs specifically expressed diphtheria toxin, causing most PCs to be knocked out, but this did not affect the proliferation of CBC cells in the crypt[16]. Later, with the identification of the Lgr5 marker and the establishment of a crypt system in vitro, the hypothesis that PCs provide a niche for stem cells was re-established. In vitro, isolated Lgr5^hi CBC cells hardly grew into crypt bodies[17]. However, when PCs and stem cells were cultured together, the stem cells could differentiate and develop into crypt bodies. Further studies in in vivo mouse models, including the Gordon model mentioned earlier, showed that knocking out PCs resulted in the loss of Lgr5 stem cells[18]. In terms of gene expression, PCs produce not only germicidal substances but also epidermal growth factor, Wnt3 and Notch ligand Dll4 in large quantities, providing necessary conditions for PCs to become a niche[19]. In conclusion, PCs provide the necessary niche signal for Lgr5^hi CBC stem cells (Figure 1).

Figure 1 Physiological function of Paneth cells.

Regulation of the intestinal flora

PCs contain a large number of endoplasmic reticulum and Golgi complexes, which have a major role in protein secretion. The main secretions of PCs are protein polypeptides with bactericidal ability, such as the cryptdin-related sequence peptide, lysozyme, IIA secretory phospholipase A2 (secretory group IIA phospholipase A2), regenerated insulin-derived proteins REG3β and REG3γ, angiogenin 4, and ANG4[20].

Antimicrobial peptides are important effectors in the innate immune response against pathogenic microbial infection. α-Defensins are one of the earliest recognized antimicrobial peptide families. They are the main components of secretory granules in phagocytes[21]. In addition to phagocytes, epithelial cells in various mucous membranes also secrete α-defensin. Mouse PCs secrete several subtypes of defensins, which can be divided into 6 subtypes from 1 to 6 by purification analysis in vitro. Immunohistochemical analysis showed that α-defensin was specifically expressed in PCs and was secreted into the intestinal cavity in a polar manner[22]. These α-defensins, which are secreted extracellularly, are thought to have important host defense functions.

Defensins are a class of small (15-20 residues) cationic proteins rich in cysteine. They are amphoteric molecules that can bind to bacterial cell membranes and form transmembrane ion channels, destroying the integrity of the membranes and causing cell contents to leak, thus killing bacteria[23]. At the same time, defensins can inactivate a variety of bacterial toxins by combining with them in order to denature them. However, how intestinal symbiotic bacteria coexist with the abundant bactericidal defensins in the gut has always been a perplexing problem. Recent studies have shown that intestinal symbiotic bacteria usually express dephosphatase (LpxF) to remove negative charges on the surface of bacteria to resist killing by cationic antibacterial peptides such as defensins[24].

Studies have shown that α-defensin secreted by PCs plays an important role in the response to pathogen infection. Gram-positive bacteria, gram-negative bacteria, lipopolysaccharide, muramic acid, muramyl dipeptides, and lipid A all stimulate defensin secretion by PCs in the small intestine[25]. Live fungi and protozoa do not stimulate PC degranulation. When PCs in the mouse small intestine encounter pathogens or pathogen antigens, they secrete granules rich in antimicrobial peptides within a few minutes to kill pathogenic microorganisms[26]. This secretion activity is dose-dependent for the pathogens or pathogen antigens. α-Defensins account for 70% of the total antimicrobial peptide killing activity[27].

It has been found that α-defensin derived from PCs plays an important role in establishing and maintaining the balance of intestinal microecology[28]. Mouse α-defensin is synthesized as a nonactivated precursor peptide and must be cleaved by matrix metalloproteinase 7 (MMP7) to be activated[29]. Two animal models, DEFA5 transgenic mice and MMP7-deficient mice, have been studied. DEFA5 transgenic mice express α-defensin 5 (also known as HD-5), which is an α-defensin overexpression model[30]. MMP7-deficient mice do not produce active α-defensin and are a model of α-defensin deficiency in the small intestine. In both mouse models, the mRNA expression levels of PC effector factors, such as lysozyme, Defa1 and Defcr4 encoding α-defcr4, and the total number of intestinal bacteria did not change significantly, but the composition of intestinal bacteria did[31]. The proportion of Firmicutes in DEFA5 transgenic mice was significantly reduced, while the proportion of Bacteroides was significantly increased, but the opposite result was obtained in MMP7-deficient mice[32]. This suggests that changes in intestinal bacterial composition are dependent on α-defensin but α-defensin does not affect the total number of intestinal bacteria[33]. Moreover, segmented filamentous bacteria, important members of Firmicutes, were barely detected in DEFA5 transgenic mice, and the proportion and number of Th17 cells in the lamina proper were also affected. These results indicate that α-defensins derived from PCs affect the intestinal symbiotic bacterial composition and intestinal homeostasis[34].

PCs not only secrete antimicrobial peptides stored in vesicles under microbial stimulation but also regulate the production of some antimicrobial peptides upon sensing microorganisms[35]. It has been found that the presence of enterobacteria can greatly enhance REG3γ expression in PCs, the upregulation of REG3γ expression depends on the MyD88 signaling pathway of the downstream adaptor molecule of the Toll-like receptor (TLR), and the REG3γ expression is necessary to prevent microbial invasion into the host tissue. Using a model with MyD88 overexpression in PCs, researchers found that PCs directly sense microorganisms through the TLR-MyD88 pathway and activate the expression of MYD88-dependent antimicrobial peptides, such as Reg3γ[36]. These results demonstrated that MyD88 signaling pathway activation in PCs is sufficient to prevent microbial invasion of the host and does not require MyD88 signaling from other sources, such as bone marrow cells[37]. This study further employed a mouse model with a defensin promoter regulating the expression of diphtheria toxin (CR2-tox176) to deplete PCs and showed that mice with PC depletion did not effectively control intestinal symbiotic and pathogenic bacterial invasion into the spleen and mucosa-associated lymph nodes. Thus, the antibacterial substances derived from PCs are very important for controlling the invasion and diffusion of microorganisms in vivo[38].

SUSCEPTIBILITY GENES

The major susceptibility gene on chromosome 16

Nucleotide-binding oligomeric domain protein 2 (NOD2)/CARD15, the first discovered C and D susceptibility gene, is located around the centromeres of chromosome 16 (16q12) and is mainly expressed in macrophages and PCs specific to the small intestinal gland. It encodes 2 CARD domains and 6 LRR (1 eukaryotic repeat) proteins. CARD15 protein activates NF-κB by recognizing the muramyl dipeptide (MDP) of foreign bacteria and plays a role in the immune response to bacterial LPS[39]. In addition, CARD15 can induce the expression of human β-defensin-2 (HBD-2) in epithelial cells when encountering invading microorganisms, which constitutes the first line of rapid defense of epithelial cells against foreign microorganisms[40]. Therefore, mutation of the CARD15 gene and subsequent alteration of the structure of the encoded protein is a risk factor for CD[41]. Most studies suggest that NOD2/CARD15 is closely related to genetic susceptibility to CD but not to UC. However, the presence of TL4 or CD14 gene mutations in conjunction with NOD2/CARD15 mutations increases UC susceptibility. The genes in IBD1 near D16s408 are also associated with the incidence of UC. For example, single allelic mutations increase the incidence of UC, while double allelic mutations can lead to severe CD[42]. Therefore, although not as significantly as with C and D, the NOD2 gene is also associated with UC.

The major susceptibility gene on chromosome 12

The human leucine-rich repetitive kinase 2 (LRRK2) gene consists of 51 exons located on chromosome 12q12 and encodes the LRRK2 protein, a multidomain protein composed of 2527 amino acids. LRRK2 is a multidomain protein kinase with a wide range of functions, including vesicle transport and entosis, protein synthesis, immune response regulation, and inflammation[43]. The LRRK2 protein consists of an ARM repeat, ankyrin repeat (ANK), leucine-rich repeat region (LRR), Ras protein complex (ROC), Ras protein C-terminal repeat (COR), kinase domain (MAPKKK) and tryptophan aspartic acid repeat region (WD40)[44]. Three scaffold domains, ANK, LRR and WD40, are involved in interactions with other proteins that can maintain the conformation and stability of those proteins. The ROC domain and MAPKKK domain are related to the GTPase activity and kinase activity of LRRK2, respectively, while the functions of the ARM repeat sequence and COR domain are not clear. Y1699C and R1628P mutations in the ROC domain have been found to be associated with Parkinson disease (PD) and leprosy, respectively[45]. Pathological and physiological studies of LRRK2 have indicated that the LRRK2 domain is significantly related to its various cellular functions, suggesting that LRRK2 is a pivotal protein with a wide range of functions. Gain-of-function mutations in the LRRK2 kinase domain lead to increased LRRK2 kinase activity and play an important role in disease pathogenesis[46].

TL1A gene

Most members of the tumor necrosis factor (TNF)/tumor necrosis factor receptor superfamily proteins (TNFR SFP) are expressed in immune cells and play a key role in the immune response[47]. Tumor necrosis factor ligand 1A (TL1A), a member of the TNFSF family, is the encoding product of the TNFSF 15 gene, and its expression is increased in the intestinal inflammatory region of patients with IBD[48]. TL1A protein has been found to be expressed in mononuclear macrophages and CD4+/CD8+ lymphocytes in the intestinal lamina propria in patients with CD, and the expression level and the number of positive cells were positively correlated with the severity of intestinal lesions[49]. Furthermore, the number of DR3-positive T lymphocytes increased in the intestinal lamina propria of CD patients. The uniform upregulation of TL1A and DR3 expression indicates that downstream cytokines after TL1A and DR3 binding play an important role in CD[50]. In addition, TL1A helps balance promotion and inhibition of the inflammatory response in the intestinal mucosa in CD. At the initial stage of inflammation, when T cells are recruited to the inflammatory site of intestinal mucosa, TL1A interacts with DR3 to enhance inflammatory cytokine secretion, and these cytokines cause the recruitment and activation of macrophages and neutrophils, stimulating further inflammation[51].

ATG16L1 gene

The Atg16L1 gene, which is involved in autophagy, is related to CD development and plays an important role in PCs, suggesting the importance of autophagy to the normal physiological function of PCs. CD patients with Atg16L1 mutations have an altered gut microbiota and abnormal PC granules[52]. A similar phenomenon was observed in mice with low expression of Atg16L1 protein. Notably, Zhang et al[53] found that autophagy of PCs was specifically activated in some CD patients, and this state was not related to mucosal inflammation and Atg16L1 mutation. This result suggested that in addition to Atg16L1, more autophagy-related genes might be involved in the pathological mechanism of CD[54]. Moreover, we do not currently know which genes are involved. Additionally, autophagy and endoplasmic reticulum stress have compensatory effects in PCs. When Atg16L1 and Xbp1 were knocked out simultaneously in the intestinal epithelium, mice developed more severe idiopathic enteritis than when either gene was knocked out alone[55].

PHYSIOLOGICAL FUNCTION OF INTESTINAL MICROECOLOGY

Changes in intestinal microecology are involved in IBD pathogenesis and development. Intestinal microecology includes intestinal microbes, intestinal epithelial cells and immune cells, among which intestinal microbes play the most important role in intestinal microecology[56]. Intestinal microorganisms are distributed on the surface of the intestinal cavity and are mainly composed of bacteria, viruses, fungi and parasites, among which the number of bacteria is approximately 10¹⁴, approximately 10 times the number of human cells[57]. The total weight of intestinal bacteria is approximately 0.2 kg, accounting for 60% of the dry weight of stool. There are more than 50 bacterial groups and approximately 1100 species, most of which are Bacteroides and Firmicutes (90%), while a small portion are Actinobacteria and Proteus[58]. Many factors have been found to influence the composition of gut microbes. At birth, the environment can directly affect the intestinal microflora, including the birth canal, early diet, antibiotic use, pet contact, sex, and the mother's health, all of which are related to the intestinal microflora composition of infants in the early period[59]. The intestinal microbial diversity of infants under 1 year of age increases rapidly and tends to stabilize at 3 years of age, the intestinal microbial composition becomes more stable at 5 years of age, and Bacteroides dominates. Adult exposure to various environmental factors (such as smoking, air pollution, hygiene habits, stress, diet, drugs, etc.) can change the intestinal microbial composition[60].

Evidence suggests that the intestinal barrier plays an important role in intestinal microbial maintenance. The intestinal barrier is composed of intestinal symbiotic bacteria, the intestinal mucous layer, the intestinal epithelium and various lymphocytes in the lamina propria[61]. The intestinal mucus layer covers the intestinal epithelium, and its components are secreted by intestinal epithelial cells; this layer act as a physical barrier between the flora and the intestinal epithelium and provides nutrients and a living environment for the intestinal flora[62]. The mucus layer is rich in mucus secreted by goblet cells, a variety of antibacterial substances secreted by PCs and ordinary epithelial cells, and IgA secreted by B cells, which are difficult obstacles for intestinal bacteria to cross, effectively preventing contact with and invasion of intestinal epithelium by intestinal bacteria and preventing inflammation[63]. Intestinal epithelial cells include goblet cells, PCs, M cells, neuroendocrine cells and absorptive intestinal epithelial cells, and intestinal epithelial cells are mainly connected by tight junctions, which can prevent the invasion of bacteria and their derivatives[64]. Moreover, there are a variety of gut-associated lymphoid tissues in the intestinal epithelium and lamina propria, such as Peyer's patches in the small intestine and lymphatic follicles and colonic patches in the large intestine[65]. Many immune cells, such as dendritic cells, T lymphocytes and B lymphocytes, exist in these enteric-associated lymphoid tissues. These lymphocytes cooperate with each other to promote immune tolerance and participate in host defense. Among them, M cells and dendritic cells directly sense intestinal contents and transmit information about the intestinal flora to other immune cells, inducing an immune response or tolerance[66].

INTERACTION OF PCS WITH INTESTINAL MICROECOLOGY

PCs regulate intestinal microecology and intestinal epithelial regeneration and differentiation

Normally, PC secretions are slowly released, and degranulation can be caused by feeding, microbial stimulation, and M receptor agonists[67]. There are many bacteria or foreign bacteria in the lumen. PCs can directly detect bacteria and express large quantities of particulate matter containing antibacterial factors through TLRs, which induces degranulation to increase the concentration of antibacterial factors in the intestinal cavity, inhibit the invasion of exogenous microorganisms, and control the microbial population in the small intestine[68]. This is an important reason why the microbial colonization density in the small intestine is lower than that in the large intestine. Therefore, PCs play important roles in controlling the passage of symbiotic bacteria and pathogenic bacteria through the intestinal barrier and in maintaining host and microbiological stability on the mucosal surface[69].

PCs and crypt basal columnar stem cells together constitute the stem cell microenvironment. The epithelial growth factors Wnt3 and Notch act on intestinal stem cells, which can promote self-renewal of intestinal stem cells and immune differentiation of different intestinal cell lines[70]. Therefore, PCs can regulate intestinal microecology and maintain intestinal mucosal homeostasis through mucosal defense and regulation of intestinal epithelial differentiation[71].

The distribution and maturation of PCs depend on intestinal microecology

Studies have shown that the distribution of intestinal bacteria in ordinary wild mice, laboratory mice and specific pathogen-free mice decreased successively, and the distribution of PCs in the same part of the small intestine of mice among these three populations also decreased successively, suggesting that the reduction in the number of bacteria in a certain part of the small intestine could lead to a decrease in the number of PCs in that part of the small intestine[72]. After consumption of amoxicillin for 3 d, the number of PCs in each segment of the small intestine decreased significantly, which may be because amoxicillin, as a broad-spectrum antibacterial, can kill a large number of gram-positive and gram-negative bacilli in the intestine, resulting in a decrease in the total number of bacteria in each segment of the small intestine and then the number of PCs in each segment of the small intestine[73]. However, after 1 d of amoxicillin consumption, the number of PCs in the jejunum and ileum increased significantly, which may be related to the sudden disturbance of intestinal microecology leading to a temporary increase in the number of PCs in a certain period of time, which improves the body's defense function[74].

In normal mice at 4 to 6 wk of age, there is a certain degree of particle abnormality in PCs, but with increasing age, the number of normal PCs gradually increases, while the number of abnormal PCs gradually decreases[75]. However, there is no obvious PC proliferation and maturation with age in germ-free mice. Compared with that in conditions without specific pathogens, the average number of PCs per crypt is lower under completely sterile conditions, and the proportion of the normal form of PCs was almost zero in young and old mice[76]. This finding suggests that the number, morphology, and maturation of normal PCs are dependent on the intestinal flora[77].

Changes in intestinal microecology have an obvious effect on the number of PCs in the small intestine of mice[78]. After Escherichia coli (E. coli) infection, the number of PCs in each segment of the small intestine of mice is significantly reduced, which may be related to intestinal damage caused by E. coli infection[79]. Studies have shown that the intestinal villi of mice infected with E. coli have varying degrees of damage, and the longer the infection extends, the more serious the intestinal villus damage, especially duodenum damage, which is the most serious, with thinning of some parts of the intestinal wall and intestinal inflammatory cell infiltration[80]. In addition, the decrease in the number of PCs after E. coli infection may be related to the large number of released particles participating in the intestinal inflammatory response[81]. A significant decrease in the number of PCs will lead to a decrease in the barrier defense function of the intestinal tract, resulting in further damage to the small intestine of mice by opportunistic pathogens or infecting E. coli[82]. Therefore, with the extension of infection time, the number of PCs is significantly reduced. Thus, the number of PCs is closely related to the intestinal microecological balance.

PANETH CELL ABNORMALITIES AND MICROECOLOGICAL DISORDERS IN IBD

PC abnormalities in IBD patients and mouse models

PCs are an important component of the intestinal epithelial barrier, so it is not surprising that functional abnormalities of PCs play a role in CD development[83]. The first pathological analysis of small intestine samples from CD patients revealed the presence of intracellular vesicle abnormalities in PCs in these patients[84]. A better understanding of the role of PCs in CD development resulted from the discovery of CD susceptibility genes. A study showed that a CD susceptibility gene was highly expressed in PCs, which supported PCs as the origin of the disease[85]. Recently, it has been found that many CD susceptibility genes are involved in the important physiological activities of PCs, and research on the pathways regulated by these genes has revealed the significance of these pathways in regulating the physiological activities of PCs[86].

The abnormalities of PCs in IBD are mainly reflected in the abnormal quantity, morphology and function of PCs and their secretory granules[87]. Upon morphological examination of PCs from CD patients, abnormal PCs were found in 20%-50% of patients. Changes in susceptibility genes or risk-associated polymorphic sites can also cause PC abnormalities, and the more susceptibility genes CD patients carry, the higher the proportion of abnormal PCs[88]. For example, mutations in the CD susceptibility genes Atg16 L1 and Xbp1 resulted in abnormal particle morphology and a reduced number of PC particles in genetically deficient mice and in CD patients. Abnormal particle morphology and antimicrobial protein packaging in PCs were also observed in engineered CD-associated autophagy protein-deficient mice[89].

Microecological dysregulation in IBD patients and mouse models

Many studies have shown that microecological disorders exist in the intestinal tract of both IBD patients and mouse models[90]. Obvious PC defects can be seen in children with CD, and abnormal PCs can cause an increased abundance of inflammatory bacteria (Corynebacterium, Erysipelotrichaceae, etc.) and reduced abundance of barrier bacteria (Faecalibacterium, Blautia, etc.). Prevotella was found to be significantly enriched in IBD patients with susceptibility genes, which may lead to a loss of intestinal barrier function and in turn to increased epithelial cell penetration and chronic inflammation[91].

PC ABNORMALITIES MEDIATED BY SUSCEPTIBILITY GENES AND MICROECOLOGICAL DISORDERS PROMOTE THE DEVELOPMENT OF INTESTINAL INFLAMMATION

Effects of the NOD2 gene on PCs and intestinal inflammation

Nod2 is the most significant CD susceptibility gene. In macrophages, NOD2 recognizes bacterial-derived muramyl dipeptides and activates the immune response[92]. Three major Nod2 mutants (R702W, G908R and L1007insC) have been found to be associated with the development of CD, resulting in the inability of NOD2 to effectively activate the downstream immune response[93]. In NOD2-deficient mice, α-defensin expression in PCs is decreased, terminal ileal symbiosis was increased, and pathogenic bacteria were enriched. In particular, granulomatous inflammation characterized by increased expression of Th1-related genes and inflammatory cytokines was observed in the ileum of NOD2-deficient mice inoculated with Helicobacter hepatis[94]. However, the overexpression of α-defensin in mouse PCs via transgenic technology resulted in decreased Th1 inflammation. Therefore, NOD2 can effectively inhibit the development of Th1-induced ileal granulomatous inflammation in mice. It is currently believed that lysozyme sorting in PCs is carried out through a NOD2-LRRK2-RIP2-Rab2A pathway dependent on intestinal symbiotic bacterial stimulation[95]. The absence of any one of NOD2, LRRK2, RIP2, Rab2A or symbiotic bacteria will cause lysozyme in PCs to be degraded by lysosomes, resulting in the breakdown of the balance between the organism and symbiotic bacteria, thus promoting the occurrence of CD[96].

However, the specific role of Nod2 mutations in the development of inflammatory enteritis is still under investigation. Because all three Nod2 mutations reduce the ability of Nod2 to activate the immune response, Nod2^-/-mice are widely used to study the role of NOD2 in inflammatory enteritis[97]. Nod2 is mainly expressed in PCs and bone marrow-derived lymphocytes in the small intestine. The downregulation of α-defensin expression in Nod2^-/-mouse PCs was reported to result in a decreased immune response to listeria[98]. It has been found that α-defensin expression in PCs is significantly reduced in patients with ileum CD compared with that in healthy individuals or patients with other types of IBD[99]. The decrease in α-defensin levels in PCs is not affected by intestinal inflammation, suggesting that the decrease in α-defensin levels is not caused by inflammation but is probably an inherent phenomenon that occurs early[100]. There was also a decrease in α-defensin levels in CD patients with 1007fs (SNP13) NOD2 mutations. However, some studies have not been able to identify defects in α-defensin production in Nod2^-/- mice, which may be related to the genetic background of the mice[101]. Additional studies have shown that Nod2 functions by regulating the intestinal flora, as Nod2^-/- mice have an altered intestinal flora, and Nod2^-/- mice have granulomatous lesions in the ileum after infection with Helicobacter pylori, consistent with the pathological state of CD[102]. This granulomatous damage is relieved when PCs of Nod2^-/- mice are transferred to α-defensin HD5 gene knockout mice.

Effects of LRRK2 gene deficiency on PCs and intestinal inflammation

TLRs in PCs can activate and promote the formation and secretion of antibacterial particles such as lysozyme after directly sensing intestinal bacteria and their metabolites. NOD2 secreted by PCs senses the presence of symbiotic bacteria by detecting the cell wall acyl dipeptides of symbiotic bacteria, and symbiotic microbiota-derived signals trigger NOD2 binding to receptor interaction protein 2 (RIP2)[103]. LRRK2 and Rab2a are recruited into dense core vesicles (DCVs) containing lysozyme to regulate the sorting of lysozyme in DCVs[104]. In this process, on the one hand, LRRK2 can affect the composition of intestinal microorganisms by regulating lysozyme sorting. On the other hand, symbiotic flora constituents can not only guide lysozyme sorting in PCs but also promote symbiosis between the symbiotic flora and host through the NOD2-LRRK2-Rab2a axis[105].

Cellular life activities depend on the precise transfer and directional transport and secretion of intracellular substance transport systems. If the regulation of the vesicle transport system is abnormal, the normal life activities of cells will be affected[106]. Abnormal PC vesicle transport, characterized by a reduced number of DCVs containing lysozyme, occurs in CD patients[107]. A similar phenomenon was also observed in LRRK2^-/- mice. Although the expression level of lysozyme mRNA in the PCs of LRRK2^-/- mice was normal, lysozyme deficiency was also found in the intestinal cavity because lysozyme was degraded by intracellular lysosomes[108]. However, other antibacterial substances, such as defensin and islet RegIIIγ, in PCs were not affected by LRRK2 knockout, suggesting that LRRK2 can specifically regulate lysozyme transport and secretion in PCs[109].

CD patients often exhibit significant dysregulation of innate immunity in the intestine. In CD pathogenesis, cytokines such as interferon-γ (IFN-γ), IFN-β, TNF-α and IL-6 can induce and upregulate LRRK2 expression[109]. Additional studies have shown that LRRK2 is involved in inflammatory cytokine production and macrophage chemotaxis in the innate immune response. LRRK2 mutation may regulate the innate immune response in CD14+ monocytes[110]. Therefore, abnormal LRRK2 expression may aggravate the innate immune disorder and further damage the tissue.

The NOD(1/2)/RIP2 signaling pathway is an important signaling pathway for the innate immune response to bacterial infection and endoplasmic reticulum stress[111]. LRRK2 enhances the activity of RIP2 by promoting the phosphorylation of RIP2 at Ser176, thus enhancing NOD(1/2)/RIP2 signaling and promoting the production of inflammatory cytokines[112]. Because LRRK2 overexpression activates NF-κB and promotes inflammatory cytokine secretion in lamina propria dendritic cells in mice, an LRRK2 overexpression group exhibited more severe colitis symptoms in DSS-induced colitis mice than a control group (wild-type mice)[113]. LRRK2 inhibitors can reduce LP-induced TNF receptor-related factor 6 (TRAF6) interaction with LRRK2 and inhibit MAPK and NF-κB suppressor protein α (Ik Bα) phosphorylation by inhibiting LLP-induced kinase activity of the LRRK2 protein[114]. Thus, the production of the inflammatory cytokine TNF-α in the dendritic cells of CD patients is reduced, which exerts an anti-inflammatory activity and ameliorates the symptoms of DSS-induced colitis[115]. In addition, a similar phenomenon has been found in macrophages, where LRRK2 defects significantly inhibit the secretion of inflammatory cytokines by macrophages when NOD2 is activated by muramyl dipeptide and NOD1 is activated by γ-D-glutamine-racemo-disaminophenic acid (I-e-DAP) or endoplasmic reticulum stress. In conclusion, LRRK2 can positively regulate the secretion of inflammatory cytokines[116].

However, other studies have shown that LRRK2 has the opposite regulatory effect. Activated T nuclear factor (NFAT) is an important mediator in the immune response, while LRRK2 is a major component of the NRON complex, an inhibitor of NFAT[117]. Normally, LRRK2 can trap NFAT by forming NRON complexes in the cytoplasm, thus sequestering NFAT in the cytoplasm[118]. However, in LRRK2^-/- mice, LRRK2 deficiency leads to the failure of NFAT sequestration in the cytoplasm, thus causing NFAT translocation to the nucleus and inducing the transcription of inflammatory cytokines, increasing the level of inflammatory cytokines, increasing susceptibility to DSS-induced colitis and aggravating colitis symptoms[119]. This finding suggests that LRRK2 may also negatively regulate the secretion of inflammatory mediators and cytokines by sequestering NFAT in the cytoplasm.

When LRRK2 is defective or mutated, NFAT regulation is altered, which activates the expression of inflammatory genes in macrophages, exacerbating intestinal inflammation in colitis mouse models and leading to the development of CD[120]. It has also been found that the lysozyme mRNA expression level in the PCs of LRRK2^-/- mice was normal, but lysozyme is readily degraded by intracellular lysosomes, while other antibacterial substances in PCs are not affected by LRRK2 deletion, suggesting that LRRK2 regulates lysozyme transport and secretion in PCs[121].

Effects of XBP1 gene deficiency on PCs and intestinal inflammation

Another remarkable genetic factor of the CD unfolded protein response (UPR) is the transcription factor X-box binding protein-1 (XBP-1), which is a key transcription factor in the endoplasmic reticulum stress response and is involved in UPR regulation, endoplasmic reticulum amplification, and the development of hypersecretory cells (such as PCs)[122]. XBP-1 can regulate the number of PCs by preventing apoptosis and mediating cell renewal. Furthermore, mucosal defense function and susceptibility to IBD are affected[123]. The specific clearance of XBP1 from IECs induces endoplasmic reticulum stress, PC loss, reduced lysozyme and defensin expression, increased IEC death and idiopathic enteritis. Similarly, PC-specific clearance of XBP1 can produce similar symptoms of spontaneous ileitis, suggesting that the PC-specific UPR plays an important role in maintaining ileal mucosal homeostasis in mice[124]. Xbp1 knockout in the mouse epithelium resulted in spontaneous enteritis in mice[125]. Pathological analysis showed that the deletion of Xbp1 resulted in endoplasmic reticulum stress and a lack of lysozyme and α-defensin expression in PCs[125]. Therefore, XBP1 may play a partially compensatory role in inhibiting proinflammatory signals, maintaining mucosal homeostasis and assisting PC function in mice, thus supporting the function of PCs[126].

Effects of TL1A overexpression on PCs and intestinal inflammation

TL1A/DR3 (TL1A functional receptor) signal transduction can not only promote the proliferation of T-effector cell subsets but also promote the production of cytokines and accelerate the progression of inflammatory diseases[127]. Different levels of TL1A expression in mice have different effects on PCs and intestinal inflammation. With increasing age, the number of PCs in wild-type mice gradually increased, lysozyme particles gradually matured, and there was no spontaneous ileitis[128]. Although the number of PCs in TL1A^-/- mice was reduced, normal lysozyme particle morphology was maintained, so spontaneous ileitis did not occur in older TL1A^-/- mice[129]. However, in the TL1A-overexpressing mouse model, the number of PCs increased significantly, and the granules did not mature with age, accompanied by spontaneous ileitis and long-distance intestinal stenosis[130]. However, an anti-TL1A antibody could block TL1A function, reverse colonic fibrosis, and reduce existing colonic inflammation. Therefore, overexpression of TL1A can aggravate proximal intestinal inflammation and fibrous stenosis and promote disease progression[131].

Effects of the ATG16L1 gene on PCs and intestinal inflammation

ATG16L1 is an autophagy-related protein that protects against PC necrosis by participating in autophagosome formation, maintaining autophagy and mitochondrial homeostasis, and preventing PC necrotic apoptosis mediated by TNF-α[132]. In addition, ATG16L1 can also play an important role in the pathogenesis of CD by affecting the extracellular secretion of PC particles in patients or activating the transcription factor XBP1 in the endoplasmic reticulum stress response[133]. In IEC-specific ATG16L1 knockout mice, loss of autophagy resulted in increased IEC sensitivity to TNF-induced cell death. Moreover, since defensins and antimicrobial peptides are mainly secreted by PCs, changes in the abundance distribution of protein components in PCs caused by ATG16L1 defects may also trigger CD[134].

ATG16L1 T300A is the most important risk-associated polymorphic site of ATG16L1. Abnormal particle morphology and antibacterial protein packaging in PCs can be seen in mice injected with ATG16L1 T300A or in CD patients carrying ATG16L1 T300A[133]. Lysozyme in PCs is packaged and secreted in secretory autophagy during bacterial infection, and secretory autophagy was inhibited in mice carrying ATG16L1 T300A in PCs[133]. In addition, ATG16L1 T300A can also reduce selective autophagy, shorten the remission interval, increase cytokine release and reduce intracellular bacterial clearance, leading to abnormal PCs, early immune infiltration and intestinal ecological disorders[134] (Figure 2).

Figure 2 Paneth cells and intestinal inflammation. TNF: Tumor necrosis factor.

CONCLUSION

IBD pathogenesis is influenced by genetics, the environment, the intestinal flora and immunity, among which abnormal PCs play a central role. Both susceptibility genes and their risk-associated polymorphic loci can cause the development of abnormal PCs, and the more susceptibility genes a patient carries, the higher the proportion of abnormal PCs. The interaction between abnormal PCs and intestinal microecology is reflected in two aspects. On the one hand, the stability of the intestinal microecology needs to be maintained by the physiological function of PCs, and abnormal PCs can also cause an imbalance in intestinal microecology. On the other hand, the proliferation and maturation of PCs depend on intestinal microecology, and environmental conditions can also aggravate the influence of susceptibility genes on PCs by changing the intestinal microecology. Intestinal microecology and susceptibility genes interact with each other. On the one hand, intestinal microecology can enhance the effect of susceptibility gene expression products and promote the occurrence of IBD. If there is no intestinal flora, abnormal susceptibility genes cannot cause idiopathic enteritis. On the other hand, when IBD-related genes are abnormal, the abundance of specific bacteria in the gut is altered, which can disrupt the intestinal barrier and promote chronic inflammation. In conclusion, susceptibility genes may cause PC abnormalities and intestinal microecological disorders, and the interaction among the three can lead to potential diseases or aggravate existing diseases when they reach a specific functional threshold. In the future, more clinical and disease mechanism studies are needed to identify other genes associated with PC abnormalities and explore the cellular and molecular mechanisms of PC abnormalities caused by susceptibility genes. In addition, exploring ways to restore intestinal homeostasis by regulating the intestinal microecosystem, improving diseases by utilizing the mutual benefit between the host and intestinal flora, and identifying peripheral markers related to CD development and PC activity changes can also provide new methods for the diagnosis, treatment and prognosis of CD.