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intestine, Wnt and Notch signaling path- The role of autophagy in ways are dominant. Th ese pathways are additionally important in maintaining Paneth cell differentiation the crypt structure and proliferation of stem/ progenitor cells. Sox-9 is one of the and secretion key transcription factors that acts down- stream of Wnt signaling to specify Paneth T S S t a p p e n b e c k 1 cells, as knockout of this gene leads to mice with crypts and active stem cells, but no Paneth cells. Other transcription Paneth cells are a small intestinal epithelial cell lineage that is factors such as the ets factor, Spdef, which considered to have a role in innate immune function. Recent interact with both Wnt and Notch signal- studies on mice with diminished and/ or loss of autophagy have ing pathways, are required for Paneth cell suggested that Paneth cells are a primary target in vivo . diff erentiation. Th e location of Paneth cells in the crypt Interestingly, loss of autophagy affects the secretion of base is likely to be strategically impor- proteins from Paneth cells. Understanding the tant to their highly specialized function, intersection of the autophagy pathway with the secretory which is to produce and secrete specifi ed apparatus, which is a key feature of differentiation of Paneth cells, proteins. In the , Paneth is a key unanswered question. cells are a critical source of cationic anti- microbial proteins that are delivered to the lumen of the gut by a process of merocrine secretion through their apical Paneth cells are a morphologically and of the other three diff erentiated lineages plasma membrane. Th is aspect of secre- geographically distinctive epithelial cell that are produced by stem cells: absorp- tion is by far the most highly studied in type located throughout the length of the tive , -producing goblet these cells. 3 Interest in this process has small intestine in both mice and humans. cells, and hormone-secreting enteroen- been driven by histological observations Another location for these cells is the docrine cells. Th ese three lineages emerge fi rst made in the late 19th century that proximal human colon that occurs only from crypts and migrate onto evagina- showed that these cells have very distinc- as a metaplastic response to an infl am- tions of the mucosal surface into the tive cytoplasmic granules. Th ese granules matory insult. In the small intestine, lumen of the intestine called villus. Each can be highlighted on sections of small Paneth cells are one of the four lineages crypt supplies these cells to multiple sur- intestine prepared for light microscopy produced continuously by epithelial rounding villi (each villus is supplied by that are stained with either periodic acid stem cells. 1 Th ese multipotent stem cells 6 – 8 surrounding crypts). Th e lifespan of Shiff or various lectins including Ulex comingle with Paneth cells in the base of villus epithelial cells is relatively short (on europaeus agglutinin 1. In addition, sec- frequent epithelial invaginations called the order of 3 – 5 days) as compared with tions prepared for transmission electron crypts of Lieberkü hn. Only a few dozen Paneth cells that migrate to the base of microscopy show the well-described elec- Paneth cells populate each crypt. How- crypts where they live much longer (>30 tron-dense secretory granules (Figure 1). ever, because of the substantial number days). Biochemical and immunohistochemical of small intestinal crypts in a mouse (~ 1.1 Recent advances have been made in studies have shown that these secretory million), the overall number of Paneth the identifi cation of transcription fac- granules store various small molecular cells is >20 million. tors and signaling pathways that infl u- weight antimicrobial proteins, includ- Th e localization of Paneth cells within ence the Paneth cell lineage specifi cation ing a variety of  - (cryptdins), the epithelial monolayer of the small and differentiation (see Stappenbeck 2 angiogenin-4, , secretory phos- intestine is distinct compared with that for review). In the adult mouse small pholipase A2, and Reg3 . Similar to most

1Department of Pathology and Immunology Washington University School of Medicine, St Louis, MO, USA. Correspondence: TS Stappenbeck (stappenb@pathology. wustl.edu)

Published online 4 November 2009. doi:10.1038/mi.2009.121

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proteins to generate the classic dou- ble membrane-bound structures in the cytosol. Th ese autophagocytic vesicles are then delivered to lysosomes for recycling of individual components. In some cell types, loss of autophagy function can lead to alterations in proliferation, cell death, and loss of the ability to clear intracel- lular pathogens. Paneth cells have been observed to form autophagocytic vesicles when cells are stressed by processes such as irradiation.7 Another reason to consider the role of autophagy in Paneth cells (as well as in other intestinal cell types) is that patients with one form of infl ammatory bowel disease, Crohn ’ s disease, have polymor- phisms in specifi c genes, including the autophagy gene Atg16L1 . This pro- tein forms a complex with two other Figure 1 “Good ” Paneth cells. Transmission electron micrograph of small intestinal Paneth cells autophagy proteins, Atg5 and Atg12, from a wild-type mouse. Note the electron-dense secretory granules and layered endoplasmic reticulum. which together function to extend the membrane of a nascent autophagocytic vesicle. Loss of autophagy function in highly secretory cells, Paneth cells addi- of the mechanism of secretion of this cell fi broblasts through the manipulation of tionally contain a robust rough endoplas- type (for example, see Sato 5 ). Atg16L1 function suggests that bacterial mic reticulum and Golgi apparatus that Not only do Paneth cells potentially clearance is impaired because of loss of act as part of the “ factory ” to produce alter the composition of the microbiome autophagy.8 However, loss-of-function these proteins. in the gut lumen but these microbes can studies in mice for Atg16L1 did not fi nd The production of antimicrobial influence Paneth cell gene expression a role in the clearance of enteric patho- proteins by Paneth cells seems to have and function as well (reviewed by Stap- genic , including Salmonella and functional consequences in both health penbeck2 ). For example, one of the major Listeria.9 and disease. These antimicrobial pro- bacterial species in the small intestine, Instead, mice with loss of function for teins likely shape the composition and Bacteroides thetaiotaomicron, stimulates Atg16L1 showed defects in the secretory abundance of the species of indigenous the expression of an antimicrobial pro- function of Paneth cells. Autophagy- microbes that reside within the intestinal tein, angiogenin-4, in Paneth cells. Th is defi cient Paneth cells properly commit- lumen. Cryptdins produced by Paneth bacterium also stimulates angiogenesis in ted to diff erentiation and did not seem to cells have also been shown to have a role the mesenchymal core of small intestinal undergo premature apoptosis. However, in the clearance of pathogens. In one villi through Paneth cells. The Paneth diminished autophagy function in Paneth example, mice that were engineered to cell– intestinal bacterial cross-talk is cells led to defects in the packaging of overexpress one member of the cryptdin particularly interesting in light of recent antimicrobial proteins into granules and family of proteins were much less sus- fi ndings that show that Paneth cells can eventually their proper export into the ceptible to oral infection by Salmonella limit bacterial penetration of intestinal lumen of the gut is defective. 9 Instead typhimurium . 4 microbes across the epithelial barrier. of a robust endoplasmic reticulum and Paneth cells also secrete proteins that Th is function depends on Toll-like recep- secretory vesicles, the autophagy-defi - infl uence other functions in the intestine. tor– Myd88 signaling in Paneth cells. cient Paneth cell cytoplasm contained Th e Wnt ligands that they produce likely The fact that Paneth cells regulate few granules and was typically fi lled with aff ect stem cells in the crypt base. Th ey the ability of intestinal bacteria to pass small vesicles (Figure 2). Th e nature of also produce factors that infl uence villus through the epithelial barrier suggests these cytoplasmic vesicles is still unclear. vasculature development. Paneth cell also that autophagy may be important in this To test for the possibility that they might produce infl ammatory cytokines such as cell type. Autophagy is a process that be endocytic vesicles that sample lumi- -  (reviewed by occurs in all cells and its major function nal bacteria, we stained for bacteria on Stappenbeck 2). It is not known whether is the recycling of intracellular compo- tissue sections. However, we have thus these factors are secreted apically or baso- nents.6 Th e machinery that is required far found no evidence that these vesi- laterally. Th e ability to now grow intesti- for this macro-autophagocytic process cles contain bacteria. Microarray stud- nal crypts in culture will aid in the study is complex and requires >20 different ies of these Paneth cells showed that in

MucosalImmunology | VOLUME 3 NUMBER 1 | JANUARY 2010 9 COMMENTARIES nature publishing group

gland acinar cells, zymogenic and pari- etal cells in the , colonic goblet cells, pancreatic acinar and islet cells, and plasma cells. Each of these cells has a robust endoplasmic reticulum, Golgi, and secretory granules similar to Paneth cells. Many further studies will be required to evaluate the role of autophagy in these cell types.

DISCLOSURE The author declared no conflict of interest.

© 2010 Society for Mucosal Immunology

Figure 2 “Bad ” Paneth cells. Transmission electron micrograph of small intestinal Paneth cells REFERENCES from an Atg16L1-deficient mouse. Note the loss of electron-dense secretory granules and layered 1 . Cheng , H . & Leblond , C .P . Origin, endoplasmic reticulum that is replaced by cytoplasmic vesicles. differentiation and renewal of the four main epithelial cell types in the mouse small intestine. IV Paneth cells . Am. J. Anat. 141, 521 – 536 ( 1974 ). addition to a defect in apical secretion, limiting the expression of infl ammatory 2 . Stappenbeck , T. S . Paneth cell development, autophagy-defi cient Paneth cells showed cytokines. differentiation and function: new molecular an altered expression of infl ammatory Many questions arise from these initial cues . Gastroenterology 137 , 30 – 33 ( 2009 ). 3 . Ouellette , A . J. Paneth cells and innate cytokines such as tumor necrosis factor- studies. Why is the function of Paneth cell immunity in the crypt microenvironment .  , leptin, and adiponection. Th e major secretion targeted by loss of autophagy? Gastroenterology 113 , 1779 – 1784 ( 1997 ). signaling pathway that was enhanced What is the role of PPARs in autophagy- 4 . Salzman , N .H . , Ghosh , D . , Huttner , K . M . , Paterson , Y. & Bevins , C .L . Protection against in autophagy-defi cient Paneth cells was defi cient Paneth cells? It will be critical to enteric salmonellosis in transgenic mice PPAR (peroxisome proliferator-activated examine the role of the human suscepti- expressing a human intestinal . Nature receptor pathway). Th e role of this fam- bility allele in mouse models to further 422 , 522 – 526 ( 2003 ). 5 . Sato , T. et al. Single Lgr5 stem cells build crypt- ily of signaling pathways is thought to be study these questions. Another puzzle is villus structures in vitro without a mesenchymal anti-infl ammatory and PPAR / has a that if such a large population of Ameri- niche . Nature 459 , 262 – 265 ( 2009 ). role in Paneth cell diff erentiation. 10 Th us, cans are homozygous for the ATG16L1 6 . Levine , B . & Kroemer , G . Autophagy in the pathogenesis of disease . Cell 132 , 27 – 42 autophagy-defi cient Paneth cells, which susceptibility allele (~ 1 / 3 of the popula- ( 2008 ). have severe alterations in secretion, may tion), do all these people have defective 7 . Gorbunov , N .V . & Kiang , J. G . Up-regulation of attempt to compensate for this defect Paneth cells or is there an environmental autophagy in small intestine Paneth cells in response to total-body gamma-irradiation . by an enhancement of the expression of or second genetic hit that acts as a trig- J. Pathol. 219 , 242 – 252 ( 2009 ). components of these pathways. We also ger for defective Paneth cells? Finally, 8 . Kuballa , P. , Huett , A . , Rioux , J. D . , Daly , M .J . & found similar alterations in Paneth cells are there additional intestinal epithelial Xavier , R . J. Impaired autophagy of an intracellular pathogen induced by a Crohn’s from Crohn ’ s disease patients who were cells that, similar to Paneth cells, have disease associated ATG16L1 variant . PLoS homozygous for the known ATG16L1 defective secretion in response to loss ONE 3 , e3391 ( 2008 ). susceptibility allele, further showing of autophagy? Paneth cells are a classic 9 . Cadwell , K . et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and the relevance of this mouse model to example of a highly secretory cell that human intestinal Paneth cells . Nature 456 , human pathology. Th us, the function of produces a limited repertoire of specifi ed 259 – 263 ( 2008 ). autophagy in Paneth cells seems to have proteins for a particular function. Other 10 . Varnat , F. et al. PPARbeta/delta regulates paneth cell differentiation via controlling the a critical role in targeting antimicrobial examples of highly secretory cells in the hedgehog signaling pathway . Gastroenterology proteins to secretory granules and in include salivary 131 , 538 – 553 ( 2006 ).

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