What do paneth cells do

Glucagon-like peptide 2 GLP-2 receptor, a product of neurons, could positively regulate epithelial growth [ 51 ].

We found that the regeneration of the intestinal epithelium induced by stromal cells is mainly achieved by directly or indirectly activating the Wnt signalling pathway. Inflammatory cells such as T cells and macrophages, by produced cytokines, influence intestine regeneration. In addition, macrophages are one of the sources of Wnts in cases of intestinal radiation injury; macrophages derived from extracellular vesicles can transport Wnts to activate the Wnt pathway and promote intestinal epithelium repair and regeneration [ 55 ].

IL-4 is a cytokine that is involved in T cell differentiation and regulation of immunoglobulin production [ 56 ]. In vitro experiments found that IL-4 can inhibit the development of organoids, as manifested by the decreased proliferation of ISCs and a significant reduction in PCs [ 57 ]; the mechanism is unclear, but the authors suggest that the dysfunction of PCs may be the cause of the reduction of ISCs.

These cytokines are important for regulating intestinal homeostasis upon inflammation [ 58 , 59 ]. IL, a product of pericryptal fibroblasts, induces PC expansion via the ST2 receptor and indirectly stimulates intestinal stem cell proliferation; the Notch signalling pathway is involved in this process [ 63 ].

The role of cytokines and immune cells on intestinal epithelium is also mainly reflected on the activation of the Wnt signalling pathway, and some of these cytokines are related to PC function and number change.

The regulation of intestinal epithelium is a comprehensive and complex process. In addition to the intestinal epithelium, sub-epithelium tissues and cytokines also provide support for the steady state of the intestinal epithelium. Cell metabolism has been implicated in stem cell maintenance and differentiation in adult stem cell populations [ 65 ].

Recent studies show that nutrients play an important role in maintaining the function of ISCs. For example, enhanced cholesterol biosynthesis leads to the proliferation of ISCs [ 66 ]. Low levels of vitamin D compromise the function of ISCs [ 67 ]. The mechanistic target of rapamycin mTOR is an important nutrition sensing element. It could regulate intestinal epithelial cells and ISCs during injury conditions [ 71 , 72 ].

These processes are canonical WNT pathway and Notch pathway independent [ 7 ]. PCs can affect the proliferation and differentiation of ISCs through different metabolic pathways.

In ex vivo study, co-culture with PCs from caloric restriction mice with intestine stem cell ISCs from untreated or treated mice gave rise to more and larger secondary organoid bodies than used normal PCs. The mechanism through which PCs augment stem cell function in response to calorie restriction involves the reduction of mTORC1 signalling in the cytoplasm. Based on gene expression profiling, no changes were observed in pathways, such as Wnt or Notch, that were previously implicated in mediating the interaction between PCs and ISCs [ 76 ].

In situ glycan editing method found that high LacNAc a sugar structure, which is linked to the cell surface protein PSGL-1 P-selectin glycoprotein ligand [ 77 ] expression on the surface of Paneth cells can block LacNAc, causing ISC proliferation arrest and accompanied by Wnt and Notch target gene changes [ 78 ].

Schematic of the model for PC-mediated stem cell function. PCs augment stem cell function response to calorie restriction by reducing mTOR and supplying respiratory substrates as by-product of lactate.

In other studies, PCs affect the function of stem cells through other metabolic pathways. Treatment with glycolysis inhibitors in PCs strongly reduce their niche-supporting function and organoid formation. Ageing is an inevitable biological event and is usually accompanied by decreased function, including diminished self-renewal ability of stem cells [ 80 ].

This process involves various systems of the human body e. The molecular mechanism of ageing remains unclear. A large number of recent studies believe that chromosome instability, pro-inflammation and mTOR play an important role in the occurrence of ageing and related diseases [ 84 , 85 ]. In the intestinal epithelium, the features of ageing are accompanied by cell cycle changes, oxidative stress and enhanced apoptosis [ 86 ]. In the gut, ageing impairing the balance between stem cell reserve and differentiation [ 80 , 89 ].

In study of mouse models, the small intestine of ageing mice showed a decrease in crypt number accompanied by an increase in crypt length and width compared with those in young mice [ 88 ].

Increasing the number of terminally differentiated cells, such as PCs and goblet cells, also alters the differentiation ability of stem cells [ 88 ]. Whether PCs are involved in ageing and their effect on crypt stem cells can be validated by co-culture.

Crypts cultured from old mice yielded fewer and less complex organoids than those from young mice [ 90 ]. The same result was obtained in long-term co-culture. These phenomena indicate that PCs can affect intestinal ageing [ 90 ]. During physiological ageing, canonical Wnt signalling declined in ISCs [ 88 ]. In a similar study on old PCs, RNA sequencing showed specific deregulation of genes that encode secreted or plasma-membrane-associated proteins; however, Wnt-responsive genes and the expression of Wnt3 or EGF were not significantly altered in old PCs [ 38 ].

The extracellular Wnt inhibitor Notum was significantly upregulated in PCs from ageing mice [ 88 , 91 ]. Notum is regulated by the canonical Wnt pathway, forming a negative-feedback loop that was significantly upregulated in old PCs [ 92 ].

This signalling is also linked with ageing with an increase in mTORC1 [ 74 ]. PCs can metabolically mediate stem cell function. Metabolic changes have an unexpected effect on ageing; fasting and caloric restriction could improve the function of ISCs with or without PCs involved.

These data demonstrate that Notum produced by PCs attenuates the regenerative capacity of ageing intestinal epithelium in vivo by reducing Wnt activity in stem cells. PCs in contrast can promote the function recovery of ageing ISCs when fasting by metabolic-related pathways. Alterations in the number and function of PCs are associated with maintaining intestinal homeostasis.

Thus, any mechanism that affects PC development or function could further change the intestinal homeostasis. In mice, PCs in the intestine differentiate around postnatal day 14 [ 94 ]. In humans, PCs differentiate around week 20 of foetal gestation [ 95 , 96 ].

Wnt and Notch are two of the main signalling pathways that control the differentiation of ISCs; the differentiation of PCs is initially controlled through a Notch-dependent mechanism during secretory progenitor specification; further PC maturation is regulated by Wnt signalling [ 26 , 97 ].

The Wnt signalling pathway is vital to promote PC development and is also important for maintaining the undifferentiated state of intestinal crypt progenitor cells.

Administration of Wnt3a increases the number of PCs in intestinal organoids [ 26 ] Fig. In addition to PCs, which can secrete Wnt3, other intestinal cells, such as stromal cells, can secrete a variety of Wnt proteins [ 36 , 37 ].

The secreted Wnt proteins are transported by the multi-pass transmembrane G protein-coupled receptor Gpr and partly dependent on Rab8a-mediated anterograde transport for exocytosis [ , ].

Schematic of the model for PC regulation. Besides the Wnt signalling pathway, many other growth factors affect the development of PCs]. The PI3K-mediated suppression of Atoh1 which is required for secretory fate determination inhibits PC differentiation [ ]. Another growth factor named helix-loop-helix transcription factor MIST1 is a scaling factor; this factor can control the fate of exocrine cells such as pancreatic acinar cells, zymogenic cells of the stomach to affect secretory capacity [ ].

The Notch signalling pathway plays a significant role in controlling the cell fate of intestinal epithelial progenitor cells into absorptive and secretory lineages. The inhibition of the Notch pathway by either chemical inhibition or knockout of associated genes e. Notch1, Notch2, Dll1 and Dll4 can increase the secretory phenotype of cells, mainly goblet cells [ 97 , , , ]. The downregulation of the Notch signalling pathway promotes secretory precursors differentiating into goblet cells but may only slightly or indirectly affect the differentiation of PCs.

Progenitor cells can differentiate into PCs through downregulation of the Notch signalling pathway [ ]. A recent study showed that acute Notch inhibition leads to rapid apoptosis of PCs, while Notch activation counteracts the death of PCs caused by caspase-8 casp8 absence; these finding suggests that Notch signalling are required for PC maintenance [ 43 , ]. In addition to above signalling pathways, metabolism-related growth factors determined regulate the fate of PCs.

Ketone bodies can mediate the proliferation ability of ISCs; 3-hydroxymethylglutaryl-CoA synthase 2 Hmgcs2 is the gene encoding the rate-limiting step for ketogenesis when the deletion of Hmgcs2 leads to pronounced expansion of PCs [ 9 , ]. Tumour suppressor and kinase Lkb1 encoded by Stk11 is a bioenergetic sensor that controls cell metabolism through repression of the transcription of Atoh1, thereby restricting the differentiation of ISCs into secretory lineage [ ].

Prohibitin 1 PHB1 , a mitochondrial membrane component protein, is crucial for maintaining PCs; when loss will lead to defect of PCs, other factors, such as inflammation-associated mitochondrial dysfunction, also produce the same effect [ , ].

Inflammatory factors are also involved in the regulation of PCs. Indoleamine 2,3-dioxygenase 1 IDO1 was upregulated by inflammatory cytokines, thereby promoting the proliferation of PCs; this process blocks the activation of Notch1 [ ].

We have discussed many growth factors and inflammatory factors that can mediate the development of PCs in different angles.

The occurrence and differentiation of PCs is greatly affected by the Wnt signalling pathway but is minimally influenced by the Notch signalling pathway. In addition to traditional pathways, metabolic and inflammatory processes also affect the fate of PCs to a certain extent.

PCs are a group of specialised epithelial cells of the small intestine and contain multiple secretory granules filled with antimicrobial peptides, which are essential for control of microbial growth and maintaining intestinal integrity. Previous studies and reviews focused on how PCs shape the intestinal microbiota or response to the immune system [ , , ]. This paper provides an overview of the function of PCs and their contribution to ISC maintenance during intestinal homeostasis and injury condition.

PCs are located at the bottom of intestinal crypts. As a terminally differentiated cell, the function of ISCs are partially regulated by paracrine-specific secreted proteins Wnt, EGF or metabolic regulation under the conditions of intestinal homeostasis, including ageing and caloric restriction.

The regulation of ISCs is mainly achieved by regulating the Wnt signalling pathway, but the metabolic regulation process, which promotes stem cell function by providing metabolic substrates. In the pathological state, the function of ISCs is enhanced, and PCs can acquire stem features to repair the intestinal mucosal epithelium.

The strong plasticity of PCs is mainly achieved by reactivating the Notch signalling pathway. In the whole intestine, various cells of the intestinal lamina propria and cytokines play an important role in the steady state of ISCs and the regulation of PCs.

As mesenchymal cells, fibroblasts can promote the function of ISCs by activating the Wnt signalling pathway or secreting Wnt and EGF; this process can partially overlap with the function of PCs. Similar to stromal cells, cytokines e. The development of PCs is also regulated by a variety of growth factors and cytokines especially under pathological conditions; the differentiation of precursor stem cells into PCs is greatly affected. The main factor that effects the differentiation of PCs is the Wnt signalling pathway.

In summary, PCs can be regarded as the guardians of intestinal crypt function and have a huge regulatory effect on ISCs under pathological and physiological conditions. The interaction network between PCs and stromal cells and between PCs and differentiated intestinal epithelial cells remains unclear. Clevers H. The intestinal crypt, a prototype stem cell compartment. Basak, O. Palmer, C. Development of the human infant intestinal microbiota. PLoS Biol. Gill, S. Metagenomic analysis of the human distal gut microbiome.

Evolution of mammals and their gut microbes. Eckburg, P. Diversity of the human intestinal microbial flora. Backhed, F. Host-bacterial mutualism in the human intestine.

Microbial ecology: human gut microbes associated with obesity. Wostmann, B. The germfree animal in nutritional studies. Gustafsson, B.

The physiological importance of the colonic microflora. Commensal Host-Bacterial Relationships in the Gut. How host-microbial interactions shape the nutrient environment of the mammalian intestine.

Falk, P. Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology. Bouskra, D. Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Macpherson, A. Interactions between commensal intestinal bacteria and the immune system.

Hapfelmeier, S. Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses.

Putsep, K. Germ-free and colonized mice generate the same products from enteric prodefensins. Ayabe, T. Angiogenins: a new class of microbicidal proteins involved in innate immunity. Cash, H. Symbiotic bacteria direct expression of an intestinal bactericidal lectin.

Gaboriau-Routhiau, V. The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses. Talham, G. Segmented filamentous bacteria are potent stimuli of a physiologically normal state of the murine gut mucosal immune system. Umesaki, Y. Differential roles of segmented filamentous bacteria and clostridia in development of the intestinal immune system. Heczko, U. Segmented filamentous bacteria prevent colonization of enteropathogenic Escherichia coli in rabbits.

Induction of intestinal Th17 cells by segmented filamentous bacteria. Barthel, M. Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host. Garner, C. Perturbation of the small intestine microbial ecology by streptomycin alters pathology in a Salmonella enterica serovar Typhimurium murine model of infection.

Wells, C. Evidence for the translocation of Enterococcus faecalis across the mouse intestinal tract. Merrell, D. The cadA gene of Vibrio cholera is induce during infection and plays a role in acid tolerance.

Antibiotic-induced perturbations of the intestinal microbiota alter host susceptibility to enteric infection. Croswell, A. Prolonged impact of antibiotics on intestinal microbial ecology and susceptibility to enteric Salmonella infection. Stecher, B. Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria. PLoS Pathog. Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits.

Turnbaugh, P. Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome. Cell Host Microbe 3 , — Dominguez-Bello, M. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Ochman, H. Evolutionary relationships of wild hominids recapitulated by gut microbial communities.

Dethlefsen, L. The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing. Peterson, D. IgA response to symbiotic bacteria as a mediator of gut homeostasis. Cell Host Microbe 2 , — Suzuki, K.

Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut. Meyer-Hoffert, U. Secreted enteric antimicrobial activity localises to the mucus surface layer. Gut 57 , — Mukherjee, S. Multi-layered regulation of intestinal antimicrobial defense. Petnicki-Ocwieja, T.

Nod2 is required for the regulation of commensal microbiota in the intestine. Satoh, Y. Carbamylcholine- and catecholamine-induced intracellular calcium dynamics of epithelial cells in mouse ileal crypts. Gastroenterology , — Stem cells, self-renewal, and differentiation in the intestinal epithelium. Barker, N. Identification of stem cells in small intestine and colon by marker gene Lgr5.

Snippert, H. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells.

Lopez-Garcia, C. Intestinal stem cell replacement follows a pattern of neutral drift. Cell Stem Cell 6 , 25—36 Development and differentiation of the intestinal epithelium. Scoville, D. Current view: intestinal stem cells and signaling. Ganz, T. Defensins: antimicrobial peptides of innate immunity. Lehrer, R. Primate defensins. Selsted, M. Mammalian defensins in the antimicrobial immune response. Functional interaction of human neutrophil peptide-1 with the cell wall precursor lipid II.

FEBS Lett. Sass, V. Schneider, T. Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II. Defensins: antimicrobial and cytotoxic peptides of mammalian cells. Yang, D. Mammalian defensins in immunity: more than just microbicidal. Trends Immunol. Lencer, W. Although Paneth cells were first discovered and described in humans, they are not specific to humans. Paneth cells can be found in many other vertebrates including primates, rodents, horses, sheep, certain fish, and chickens 5 , 6.

While Paneth cells have been found in this wide variety of other organisms aside from humans, the ontogeny and function are not well-understood for most of them aside from the well-studied and characterized rodents as well as humans. Today, Paneth cells still capture the attention of researchers as they serve an essential role in modulating the microbiome, playing a key part of the innate immune response, and aiding in the proliferation and differentiation of the intestinal epithelium.

While Paneth cells have been shown to play important roles in the healthy gut of adults, the development and role of Paneth cells in the immature gut of the preterm infant remains an understudied, but crucial avenue of research that could aid in the understanding of the development of intestinal diseases such as necrotizing enterocolitis NEC. This review sets out to unveil some of the mystery surrounding Paneth cells in the context of the preterm infant gut and how it relates to NEC.

The human gastrointestinal surface is the largest surface area of the body that is in contact with the external environment 7 , 8. This massive surface area is required to allow sufficient nutrient absorption to support growth and health of the host. The small intestine, where Paneth cells reside, has an estimated surface area of cm 2 at birth, which grows and expands to over 30 m 2 by adulthood 7 , 8.

To achieve such a massive surface area, the intestinal surface is clad by fingerlike projections that stick out into the intestinal lumen creating an expansive folding system. This systems' entire surface is covered by a single layer of columnar intestinal epithelial cells IECs. The intestinal epithelium is the most rapidly-renewing tissue in the adult mammal 9 and undergoes continuous turnover that is generated from Intestinal Stem cells ISC.

The ISC reside at or near the base of the pocket-like intestinal crypts 10 , 11 and continuously generate daughter cells that differentiate near the top of the crypts before migrating toward their final destinations. The typical pattern for these cells is to migrate upwards toward the villus tip in a conveyor-belt-type fashion until they are sloughed off the upper villus into the lumen.

However, a unique aspect of Paneth cell biology compared to the other intestinal epithelial cell types is that instead of flowing upward out of the crypt, Paneth cells move downwards further into the crypt as they mature. In addition, while most epithelial cells are rapidly turned over in a few days, Paneth cells can persist for just under 1 month Paneth cell presence is an intestinal priority and their density is rapidly repopulated following their depletion Following their descent into the crypts, Paneth cells are interspersed between the ISCs and can be distinguished by their columnar to pyramidal shape and by the presence of eosinophilic granules within their cytoplasm Figure 1.

Figure 1. The intestinal epithelium. Right Schematic of the intestinal epithelium, associated microbial flora, epithelial cell types goblet cells, Paneth cells, enterocytes, and stem cells intestinal microvasculature, and mucus layer.

Paneth cells first appear in the small intestine of humans at Paneth cell density in the developing fetal intestine is relatively low, but gradually increases throughout gestation, with significant increases in the third trimester after 29 weeks completed gestation 17 , Paneth cell levels do not reach quantities similar to adult levels until term gestation or later Because Paneth cells are located primarily in the distal small intestine, studies using human tissues have been challenging.

Thus, much of our understanding of in vivo Paneth cell biology has been generated using animal models, predominantly in mice. It is therefore important to note that not all mammals develop Paneth cells prenatally, but instead develop them mid-way through intestinal development after villus development, but before intestinal maturity according to a normal developmental pattern.

Paneth cells, like all other intestinal epithelial cell types, are derived from ISCs. In the last decade, it has become clear that ISCs are quite complex. Current models suggest multiple, potentially interconvertible populations of stem cells exist. The first is the crypt-base columnar CBC cells 20 , slender cells wedged at the very base of the crypt between the Paneth cells.

The second ISC population express Bmi1, mTert , and Lrig1 markers, and have been hypothesized to be quiescent stem cells until injury occurs, at which time they actively proliferate and produce daughter progeny Interconversion between the two compartments and overlap between the populations has been demonstrated These cells become differentiated as they migrate, and both their differentiation and the maintenance of the stem cells in their proper place is driven through gradients and juxtracrine signaling of Bmp, Wnt, Notch, and growth factor pathways 25 , Furthermore, while the exact sources of ligands for these pathways are not fully understood, it is important to note that Paneth cells produce EGF, Notch, and Wnt, which in turn promote stem cell proliferation and maintenance Several biochemical pathways have been implicated in the development of Paneth cells Figure 2.

However, the Wnt signal pathway and its relationship to Paneth cell development is complex and still not completely elucidated. Genetic knockout of LGR-5, a downstream target of Wnt signaling has been shown to produce precocious Paneth cell differentiation in fetal intestine 29 , This contradictory data may be due to alterations in negative feedback mediators in the Wnt pathway. Atoh1 has also been shown to be affected by ErbB3, a Receptor Tyrosine Kinase also known as neuregulin Genetic loss of ErbB3 in mice results in unchecked activity of the transcription factor Atoh1 and induces precocious appearance of Paneth cells In addition, activation of ErbB3 can delay normal Paneth cell development.

It is however important to note that modifications to Atoh1 signal pathways also affect goblet cell differentiation 36 , so understanding of signal pathways that distinguish goblet cell from Paneth cell differentiation downstream of Atoh1 is still incomplete. Figure 2. Intestinal epithelial cell differentiation pathways. While enterocytes further differentiate through HES-1 signaling, secretory lineages can differentiate into different cell types depending on conditions.

Wnt signal pathways drive ISC differentiation into secretory precursor cells. Secretory precursors then develop either into enteroendocrine cells through Neurog3 signaling, or into goblet and Paneth cells following activation of Atoh1. Differentiation signal pathways to separate development of goblet cells and Paneth cells are still unknown. It is also important to note that recent data has shown that activation of ErbB3 acts as a suppressor of Atoh1, while genetic deletion of ErbB3 induces precocious development of Paneth cells.

After their migration to the crypt base and subsequent maturation, Paneth cells can be easily distinguished by their prominent acidophilic granules. The granules hold many of the proteins and peptides that Paneth cells secrete to both modulate the microbiome and mediate the inflammatory response. These granular components are assembled and packaged by an extensive endoplasmic reticulum ER and Golgi apparatus network into dense core granules. IgA is one such component which may be produced by plasma cells in the lamina propria before accumulating and associating in Paneth cell granules Since Paneth cells are not currently able to be cultured without other epithelial and stem cells, most of the data we have on granular contents is from immunohistochemistry techniques.

The granules are then released at the apical surface of the cell into the lumen of the intestine where they serve a variety of biological functions, primarily as microbiocidal agents against bacteria, fungi, spirochetes, protozoa, and enveloped viruses Paneth cell granules are secreted both constitutively and in response to pathogenic exposure, with common stimuli including cholinergic stimulation and exposure to bacterial antigens 45 — This secretion of Paneth cell granular components is under tight regulatory control, as these mediators are vital for maintenance of intestinal homeostasis 38 , 48 , Paneth cell health remains a critical priority to the homeostasis of the small intestine.

We and others have shown that following dithizone-induced loss, the small intestine replenishes Paneth cell populations within 72 h 14 , 50 , Since the mammalian intestinal tract represents the largest surface area that communicates with the external environment 7 , 52 , protection of the host from injury or bacterial invasion from the intestinal flora 53 requires a complex system of defense mechanisms.

In the small intestine, a key component of host defense is epithelial derived antimicrobial peptides AMPs. These peptides are the main product contained in Paneth cell granules. In humans, there are two major classes of AMPs: cathelicidins and defensins. Cathelicidins are antimicrobial peptides with broad antibacterial 54 , anti-fungal 55 , and anti-viral activity 56 , and are characterized by a highly conserved N-terminal domain. Only after cleavage of the AMP does the protein exert its myriad activities Humans express only one cathelicidin, LL originally hCAP 58 and it is expressed in various cells of the body including those of the intestinal epithelium 59 — However, in the small intestine, cathelicidin expression is restricted to the neonatal period 63 , 64 before markedly decreasing and disappearing.

It is important to note that mid-development of the small intestine is also around the time when NEC often occurs in infants born extremely prematurely 67 Figure 3. Figure 3. Small intestinal AMP switch during intestinal development. In infants born prematurely, this switch is temporally similar to when extremely preterm infants are most susceptible to develop NEC The second class of AMP found in the small intestine are defensins.

In mice, loss of matrilysin the proteolytic enzyme needed to activate cryptdins have altered microbiomes and are more susceptible to Salmonella infections 73 — In addition, mice that have been genetically modified to express HD-5 have enhanced resistance to bacterial invasion AMPs work by inserting themselves into the bacterial membrane and forming pores, which result in the leakage of bacterial cytoplasmic content 76 — They can also degenerate bacterial cytoplasmic structures and form extracellular net-like structures, which result in bacterial trapping In animal models, AMPs have been shown to preferentially target non-commensal bacteria while sparing commensal normal flora 47 , In addition to killing pathogens, AMPs can also influence the immune system through white blood cell chemotaxis 81 , activation of dendritic cells 82 , and downregulation of immunomodulators such as cortisol 68 , Cells of the body undergo death for a multitude of reasons and through various mechanisms.

The mechanisms of cell death include apoptosis, necrosis, necroptosis, pyroptosis, and autophagy. While NEC is defined by necrosis of the intestinal tissue, many of these different cellular death pathways have been implicated in the pathogenesis of NEC. Importantly, several of these pathways are also mechanistically tied to Paneth cell biology. Apoptosis is a normal part of intestinal health that results in disassembly of the cell and, in general, tends to avoid causing inflammation During apoptosis, cells tend to retract pseudopods, condense chromatin pyknosis , undergo nuclear fragmentation and then experience blebbing of the plasma membrane This contrasts with cellular necrosis where cells experience organelle swelling, extensive vacuole formation, condensation of nuclei, and release of inflammatory cytokines in a passive or accidental manner 83 , One type of apoptosis seen in the intestinal epithelial layer is when the epithelial cells move upward from the crypt toward the tip of the villus.

Once they reach the tip, cells are sloughed into the intestinal lumen in a process called anoikis, which is a form of apoptosis There is evidence to show that apoptosis is also involved in the cell death experienced by cells in the stem cell region within the small intestinal crypts although the regulation of the process is not well-understood Apoptosis has been shown by multiple investigators to be important in development of NEC 85 — Additionally, apoptosis is directly relevant to Paneth cell biology and NEC as our lab has shown that NEC-like injury can be induced in mice by delivering diphtheria toxin to PC-DTR mice where a human diphtheria toxin receptor has been attached to the cryptdin-2 promoter of Paneth cells 14 , When these mice are exposed to diphtheria toxin, all Paneth cells expressing the construct are lysed through apoptotic pathways Another form of cell death directly related to Paneth cells is autophagy, which is a self-degradative process thought to help remove cells with misfolded or aggregated proteins or other intracellular damage Autophagy is characterized by creation of an intracellular vacuole known as the autophagosome The autophagosome is formed around damaged intracellular organelles or other selected targets.

The autophagosome is then fused with a lysosome allowing for degradation of the components within the autophagosome followed by chromatin condensation The morphologic changes that occur tend to be relatively well-regulated similar to the degree of regulation of apoptosis. Also similar to apoptosis, because the degradation of the dying cell takes place within another cell, this process tends to prevent inflammation Autophagy is also an important process for Paneth cells.

Because Paneth cells tend to live longer than most other cells of the gut and have many aggregated proteins that could be recycled by other neighboring cells, as damage and stressors to the cells occur, autophagy becomes activated When mutations occur in the autophagy pathway such as in Atg16l1, Paneth cells can become dysfunctional and ultimately trigger intestinal inflammation, which can have implications for gut health such is suggested to be the case with Crohn's disease 92 and NEC Our laboratory has also shown that autophagy may play a role in development of NEC.

Lueschow et al. Due to the higher concentration of negatively-charged phospholipids in bacterial membranes than vertebrate membranes, defensins preferentially bind to and disrupt bacterial cells, sparing the cells they are functioning to protect. Paneth cells are stimulated to secrete defensins when exposed to bacteria both Gram positive and negative types or such bacterial products as lipopolysaccharide, muramyl dipeptide and lipid A. These cells also secrete lysozyme and phospholipase A2, both of which have clear antimicrobial activity.

This battery of secretory molecules gives Paneth cells a potent arsenal against a broad spectrum of agents, including bacteria, fungi and even some enveloped viruses.