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Is the Intestinal Goblet Cell a Major Immune Cell?
Cell Host & Microbe
Previews Is the Intestinal Goblet Cell a Major Immune Cell? Malin E.V. Johansson1 and Gunnar C. Hansson1,* 1Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2014.02.014
Recent reports indicate that both the inflammasome (Wlodarska et al., 2014) and autophagy (Patel et al., 2013) pathways in goblet cells control mucin secretion, defects in which are linked to difficulties in clearing pathogenic bacteria and maintaining intestinal homeostasis and control colitis. Controlling inflammation is a major challenge, and this is especially important in the large intestine, which is home to tens of billions of bacteria. Following the discovery of the inflammasome as an activator platform for caspases, which process the inflammatory cytokine IL-1b, these complexes have attracted a lot of attention within the field of immunology. The inflammasome is constituted by one of the pyrin domain contain NLR proteins (NLRPs), one of the inflammatory caspases, and the ASC adaptor protein. When NLRP6-deficient mice were studied, they turned out to be more susceptible to colonic inflammation, which was assumed to be due to defects in wound repair and dependent on NLRP6 function in cells derived from the hematopoietic lineage. More recently, it has become clear that proteins that make up the inflammasomes are also expressed in epithelial cells, and the NLRP6 protein is highly expressed in the intestinal epithelium. However, the function of epithelium-expressed inflammasome proteins has remained enigmatic. In a recent paper published in Cell, Wlodarska et al. report that NLRP6-deficient mice are unable to clear Citrobacter rodentium infection from the colonized colon (Wlodarska et al., 2014). Unexpectedly, this effect was not due to a dysfunctional immune system or not even the lack of IL-1b or IL-18—but rather due to defects in mucus secretion by the intestinal goblet cells. The authors show that lack of NLRP6 or its other inflammasome partners, caspase 1/11 or ASC, all cause defects in the secretion of the main goblet cell product, the MUC2 mucin. The major component stored in the typical goblet cell granule is the MUC2 mucin (Figure 1). This is a large glycosylated protein that has a molecular mass
of 2.5 MDa as a monomer. MUC2 monomers are further interconnected at both the N- and C-terminal ends with disulfide bonds to form enormously large molecules. MUC2 polymers are densely packed in the goblet cell granulae and upon release MUC2 unfolds and expands >1,000 times to form net-like sheets (Ambort et al., 2012). These sheets become staggered on each other to build the inner colon mucus layer with a total thickness of about 50 mm in mice (200 mm for humans). This inner mucus layer is normally impermeable to bacteria and thus effectively separates the commensal microbiota from the single epithelial cell layer (Johansson et al., 2008). Absence or any defect in this inner mucus layer will allow bacteria to reach the epithelium in large quantities and trigger overexuberant host immune responses. When mucus secretion was assessed in mice lacking NLRP6, their goblet cells were less effective in secreting mucins and showed a less-developed inner mucus layer (Wlodarska et al., 2014). When NLRP6 mice were studied previously, they were shown to have an altered colonic bacterial ecology with increased amounts of Prevotellaceae and TM7 bacteria, leading to an increased risk of developing colon inflammation as shown by dextran sodium sulfate (DSS) treatment (Elinav et al., 2011). Interestingly, these mice also showed metabolic alterations with increased levels of fat in the liver resembling nonalcoholic fatty liver disease and obesity (HenaoMejia et al., 2012). The new knowledge that NLRP6 deficiency compromises colon goblet cell function redirects the focus of the observed metabolic and inflammatory phenotypes to the colon microbiota, their interaction with the mucus layers, and the role of mucins in forming and maintaining homeostasis in
the bacterial habitat (Wlodarska et al., 2014). This work further links the inner mucus layer of colon to colitis, as was also suggested recently in observation reported with mouse models of spontaneous colitis (Johansson et al., 2014). All these models either lacked an inner mucus layer or had altered mucus properties that allowed bacteria to penetrate to the epithelium. These mice developed more or less severe colon inflammation. Patients with ulcerative colitis also have a defective inner mucus layer (Johansson et al., 2014), suggesting that bacteria in contact with the epithelium are a strong inducer of inflammation. Stappenback and coworkers showed recently that autophagy proteins are necessary for goblet cell secretion (Patel et al., 2013). Using a villin-driven targeted deficiency of the autophagy protein Atg5 in murine intestinal crypt cells, they observed an accumulation of MUC2 granules in goblet cells. This could be linked to defective goblet cell secretion. Wlodarska et al. (2014) confirm this observation, as heterozygous Atg5 knockout mice also showed defects in goblet cell secretion. Autophagy proteins have been implicated in human inflammatory bowel diseases, and involvement of autophagy proteins in goblet cell secretion further argues for a key role of goblet cell products in controlling inflammation. Intestinal goblet cells are a part of the secretory cell linage derived from the stem cells located in the crypt bottom. During differentiation goblet cells acquire the ability to produce and store large amounts of mucus, which is evident in the upper colon crypt cells. In addition, goblet cells at the colon surface epithelium produce and secrete mucin at higher rates and are responsible for the basal mucin secretion (Johansson, 2012). This basal secretion constantly renews the
Cell Host & Microbe 15, March 12, 2014 ª2014 Elsevier Inc. 251
Cell Host & Microbe
Previews
Figure 1. A Goblet Cell Filled with MUC2 Mucin Undergoing Normal Exocystosis Mucin is green; nucleus is in blue.
inner mucus layer with an estimated turnover of about an hour. In situations where the epithelium is challenged with potential hazards, the goblet cells in the upper crypt respond with compound exocytosis (Grootjans et al., 2013). These goblet cells seem to be the same cells that are most affected by the lack of both NLRP6
and Atg5 in the work of Wlodarska et al. (2014) and Patel et al. (2013). These observations suggest that there are several types of goblet cells and that we should expect them to serve different functions. In the small intestine it was recently discovered that what is believed to be goblet cells, called GAPs, take up and present luminal antigens to the lamina propria CD11c+/CD103+ tolerogenic dendritic cells (McDole et al., 2012). The mechanism of this uptake is not understood, but it was coupled to MUC2 secretion. Also, the work by Patel et al. (2013) suggest that there is coupling between endocytosis and goblet cells secretion via the Atg5 autophagy system. Thus, the goblet cell seems to be a very intricate cell where components of the autophagy system are coupled to endocytosis and secretion and somehow also linked to inflammasome components. How these systems integrate will be the focus of future studies. How goblet cell secretion is triggered and the molecular mechanisms behind goblet cell compound exocytosis also await discovery. The results by Wlodarska et al. (2014) and Patel et al. (2013) have thus just opened a window into a vast area of unexplored cell biology. Suddenly, the intestinal goblet cells are in the midst of understanding the immune system, both forming the protective mucus and as antigen retrievers. The goblet cell and the mucus it produces is also a major mediator of the gut flora and its interaction with the host. The coming years will focus immunologists, micro-
252 Cell Host & Microbe 15, March 12, 2014 ª2014 Elsevier Inc.
biologists, and cell biologists on revealing the hidden secrets of this remarkable cell that has been unintentionally ignored for such a long time. REFERENCES Ambort, D., Johansson, M.E.V., Gustafsson, J.K., Nilsson, H., Ermund, A., Johansson, B.R., Kock, P., Hebert, H., and Hansson, G.C. (2012). Proc. Natl. Acad. Sci. USA 109, 5645–5650. Elinav, E., Strowig, T., Kau, A., Henao-Mejia, J., Thaiss, C., Booth, C., Peaper, D., Bertin, J., Eisenbarth, S., Gordon, J., and Flavell, R. (2011). Cell 145, 745–757. Grootjans, J., Hundscheild, I.H., Lenaerts, K., Boonen, B., Renes, I.B., Verheyen, F.K., Dejong, C.H., von Meyenfeldt, M.F., Beets, G.L., and Buurman, W.A. (2013). Gut 62, 250–258. Henao-Mejia, J., Elinav, E., Jin, C., Hao, L., Mehal, W.Z., Strowig, T., Thaiss, C.A., Kau, A.L., Eisenbarth, S.C., Jurczak, M.J., et al. (2012). Nature 482, 179–185. Johansson, M.E.V. (2012). PLoS ONE 7, e41009. Johansson, M.E.V., Gustafsson, J.K., HolmenLarsson, J., Jabbar, K.S., Xia, L., Xu, H., Ghishan, F.K., Carvalho, F.A., Gewirtz, A.T., Sjo¨vall, H., and Hansson, G.C. (2014). Gut 213, 281–291. Johansson, M.E.V., Phillipson, M., Petersson, J., Holm, L., Velcich, A., and Hansson, G.C. (2008). Proc. Natl. Acad. Sci. USA 105, 15064–15069. McDole, J.R., Wheeler, L.W., McDonald, K.G., Wang, B., Konjufca, V., Knoop, K.A., Newberry, R.D., and Miller, M.J. (2012). Nature 483, 345–349. Patel, K.K., Miyoshi, H., Beatty, W.L., Head, R.D., Malvin, N.P., Cadwell, K., Guan, J.L., Saitoh, T., Akira, S., Seglen, P.O., et al. (2013). EMBO J. 32, 3130–3144. Wlodarska, M., Thaiss, C.A., Nowarski, R., HenaoMejia, J., Zhang, J.-P., Brown, E.M., Frankel, G., Levy, M., Katz, M.N., Philbrick, W.M., et al. (2014). Cell 156, 1045–1059.
Previews Is the Intestinal Goblet Cell a Major Immune Cell? Malin E.V. Johansson1 and Gunnar C. Hansson1,* 1Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2014.02.014
Recent reports indicate that both the inflammasome (Wlodarska et al., 2014) and autophagy (Patel et al., 2013) pathways in goblet cells control mucin secretion, defects in which are linked to difficulties in clearing pathogenic bacteria and maintaining intestinal homeostasis and control colitis. Controlling inflammation is a major challenge, and this is especially important in the large intestine, which is home to tens of billions of bacteria. Following the discovery of the inflammasome as an activator platform for caspases, which process the inflammatory cytokine IL-1b, these complexes have attracted a lot of attention within the field of immunology. The inflammasome is constituted by one of the pyrin domain contain NLR proteins (NLRPs), one of the inflammatory caspases, and the ASC adaptor protein. When NLRP6-deficient mice were studied, they turned out to be more susceptible to colonic inflammation, which was assumed to be due to defects in wound repair and dependent on NLRP6 function in cells derived from the hematopoietic lineage. More recently, it has become clear that proteins that make up the inflammasomes are also expressed in epithelial cells, and the NLRP6 protein is highly expressed in the intestinal epithelium. However, the function of epithelium-expressed inflammasome proteins has remained enigmatic. In a recent paper published in Cell, Wlodarska et al. report that NLRP6-deficient mice are unable to clear Citrobacter rodentium infection from the colonized colon (Wlodarska et al., 2014). Unexpectedly, this effect was not due to a dysfunctional immune system or not even the lack of IL-1b or IL-18—but rather due to defects in mucus secretion by the intestinal goblet cells. The authors show that lack of NLRP6 or its other inflammasome partners, caspase 1/11 or ASC, all cause defects in the secretion of the main goblet cell product, the MUC2 mucin. The major component stored in the typical goblet cell granule is the MUC2 mucin (Figure 1). This is a large glycosylated protein that has a molecular mass
of 2.5 MDa as a monomer. MUC2 monomers are further interconnected at both the N- and C-terminal ends with disulfide bonds to form enormously large molecules. MUC2 polymers are densely packed in the goblet cell granulae and upon release MUC2 unfolds and expands >1,000 times to form net-like sheets (Ambort et al., 2012). These sheets become staggered on each other to build the inner colon mucus layer with a total thickness of about 50 mm in mice (200 mm for humans). This inner mucus layer is normally impermeable to bacteria and thus effectively separates the commensal microbiota from the single epithelial cell layer (Johansson et al., 2008). Absence or any defect in this inner mucus layer will allow bacteria to reach the epithelium in large quantities and trigger overexuberant host immune responses. When mucus secretion was assessed in mice lacking NLRP6, their goblet cells were less effective in secreting mucins and showed a less-developed inner mucus layer (Wlodarska et al., 2014). When NLRP6 mice were studied previously, they were shown to have an altered colonic bacterial ecology with increased amounts of Prevotellaceae and TM7 bacteria, leading to an increased risk of developing colon inflammation as shown by dextran sodium sulfate (DSS) treatment (Elinav et al., 2011). Interestingly, these mice also showed metabolic alterations with increased levels of fat in the liver resembling nonalcoholic fatty liver disease and obesity (HenaoMejia et al., 2012). The new knowledge that NLRP6 deficiency compromises colon goblet cell function redirects the focus of the observed metabolic and inflammatory phenotypes to the colon microbiota, their interaction with the mucus layers, and the role of mucins in forming and maintaining homeostasis in
the bacterial habitat (Wlodarska et al., 2014). This work further links the inner mucus layer of colon to colitis, as was also suggested recently in observation reported with mouse models of spontaneous colitis (Johansson et al., 2014). All these models either lacked an inner mucus layer or had altered mucus properties that allowed bacteria to penetrate to the epithelium. These mice developed more or less severe colon inflammation. Patients with ulcerative colitis also have a defective inner mucus layer (Johansson et al., 2014), suggesting that bacteria in contact with the epithelium are a strong inducer of inflammation. Stappenback and coworkers showed recently that autophagy proteins are necessary for goblet cell secretion (Patel et al., 2013). Using a villin-driven targeted deficiency of the autophagy protein Atg5 in murine intestinal crypt cells, they observed an accumulation of MUC2 granules in goblet cells. This could be linked to defective goblet cell secretion. Wlodarska et al. (2014) confirm this observation, as heterozygous Atg5 knockout mice also showed defects in goblet cell secretion. Autophagy proteins have been implicated in human inflammatory bowel diseases, and involvement of autophagy proteins in goblet cell secretion further argues for a key role of goblet cell products in controlling inflammation. Intestinal goblet cells are a part of the secretory cell linage derived from the stem cells located in the crypt bottom. During differentiation goblet cells acquire the ability to produce and store large amounts of mucus, which is evident in the upper colon crypt cells. In addition, goblet cells at the colon surface epithelium produce and secrete mucin at higher rates and are responsible for the basal mucin secretion (Johansson, 2012). This basal secretion constantly renews the
Cell Host & Microbe 15, March 12, 2014 ª2014 Elsevier Inc. 251
Cell Host & Microbe
Previews
Figure 1. A Goblet Cell Filled with MUC2 Mucin Undergoing Normal Exocystosis Mucin is green; nucleus is in blue.
inner mucus layer with an estimated turnover of about an hour. In situations where the epithelium is challenged with potential hazards, the goblet cells in the upper crypt respond with compound exocytosis (Grootjans et al., 2013). These goblet cells seem to be the same cells that are most affected by the lack of both NLRP6
and Atg5 in the work of Wlodarska et al. (2014) and Patel et al. (2013). These observations suggest that there are several types of goblet cells and that we should expect them to serve different functions. In the small intestine it was recently discovered that what is believed to be goblet cells, called GAPs, take up and present luminal antigens to the lamina propria CD11c+/CD103+ tolerogenic dendritic cells (McDole et al., 2012). The mechanism of this uptake is not understood, but it was coupled to MUC2 secretion. Also, the work by Patel et al. (2013) suggest that there is coupling between endocytosis and goblet cells secretion via the Atg5 autophagy system. Thus, the goblet cell seems to be a very intricate cell where components of the autophagy system are coupled to endocytosis and secretion and somehow also linked to inflammasome components. How these systems integrate will be the focus of future studies. How goblet cell secretion is triggered and the molecular mechanisms behind goblet cell compound exocytosis also await discovery. The results by Wlodarska et al. (2014) and Patel et al. (2013) have thus just opened a window into a vast area of unexplored cell biology. Suddenly, the intestinal goblet cells are in the midst of understanding the immune system, both forming the protective mucus and as antigen retrievers. The goblet cell and the mucus it produces is also a major mediator of the gut flora and its interaction with the host. The coming years will focus immunologists, micro-
252 Cell Host & Microbe 15, March 12, 2014 ª2014 Elsevier Inc.
biologists, and cell biologists on revealing the hidden secrets of this remarkable cell that has been unintentionally ignored for such a long time. REFERENCES Ambort, D., Johansson, M.E.V., Gustafsson, J.K., Nilsson, H., Ermund, A., Johansson, B.R., Kock, P., Hebert, H., and Hansson, G.C. (2012). Proc. Natl. Acad. Sci. USA 109, 5645–5650. Elinav, E., Strowig, T., Kau, A., Henao-Mejia, J., Thaiss, C., Booth, C., Peaper, D., Bertin, J., Eisenbarth, S., Gordon, J., and Flavell, R. (2011). Cell 145, 745–757. Grootjans, J., Hundscheild, I.H., Lenaerts, K., Boonen, B., Renes, I.B., Verheyen, F.K., Dejong, C.H., von Meyenfeldt, M.F., Beets, G.L., and Buurman, W.A. (2013). Gut 62, 250–258. Henao-Mejia, J., Elinav, E., Jin, C., Hao, L., Mehal, W.Z., Strowig, T., Thaiss, C.A., Kau, A.L., Eisenbarth, S.C., Jurczak, M.J., et al. (2012). Nature 482, 179–185. Johansson, M.E.V. (2012). PLoS ONE 7, e41009. Johansson, M.E.V., Gustafsson, J.K., HolmenLarsson, J., Jabbar, K.S., Xia, L., Xu, H., Ghishan, F.K., Carvalho, F.A., Gewirtz, A.T., Sjo¨vall, H., and Hansson, G.C. (2014). Gut 213, 281–291. Johansson, M.E.V., Phillipson, M., Petersson, J., Holm, L., Velcich, A., and Hansson, G.C. (2008). Proc. Natl. Acad. Sci. USA 105, 15064–15069. McDole, J.R., Wheeler, L.W., McDonald, K.G., Wang, B., Konjufca, V., Knoop, K.A., Newberry, R.D., and Miller, M.J. (2012). Nature 483, 345–349. Patel, K.K., Miyoshi, H., Beatty, W.L., Head, R.D., Malvin, N.P., Cadwell, K., Guan, J.L., Saitoh, T., Akira, S., Seglen, P.O., et al. (2013). EMBO J. 32, 3130–3144. Wlodarska, M., Thaiss, C.A., Nowarski, R., HenaoMejia, J., Zhang, J.-P., Brown, E.M., Frankel, G., Levy, M., Katz, M.N., Philbrick, W.M., et al. (2014). Cell 156, 1045–1059.