Cell Host & Microbe

Previews Alcohol Lowers Your (Intestinal) Inhibitions Namrata Iyer1 and Shipra Vaishnava1,* 1Molecular Microbiology and Immunology Department, Brown University, Providence, RI 02912, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2016.01.014

Alcohol causes microbiota dysbiosis and breaches intestinal integrity, resulting in liver inflammation and ultimately cirrhosis. In this issue of Cell Host & Microbe, Wang et al. (2016) demonstrate that ethanol suppresses the intestinal anti-microbial response. This enables gut bacteria to trespass to the liver and thus exacerbates the disease progression. The human gut is host to a plethora of microorganisms that colonize niches ranging from the acidic stomach to the small and large intestines. This collection of microbes acts as an accessory organ, aiding in digestion and nutrient absorption and deterring pathogenic microbes from colonizing the host. The host invests heavily in maintaining and regulating this symbiotic relationship, ensuring that these seemingly innocuous microbes are sequestered in the gut and are not allowed to access extra-intestinal tissues. To this end, the body employs a threetier security system. Level one is the mucus that covers the epithelial lining in the gut. This viscous layer acts as a barrier to keep the microbes in the gut lumen from getting too close. Level two is the arsenal of anti-microbial molecules produced by the host that ensure sterility in the vicinity of the epithelium. Last but not the least is the epithelial layer itself, which ensures that the space between the cells is sealed off by tight junction proteins to keep away any trespassing microbes. In a healthy individual, this defense strategy ensures that bacteria do not cross the intestinal epithelial barrier. The consumption of alcohol, however, tips this delicate balance. Studies with animal models and human patients suffering from alcoholic liver disease (ALD) have shown that over long periods of time, alcohol causes an imbalance in the gut and compromises the integrity of the intestinal barrier. Alcohol consumption changes the environment in the gut, leading to an overgrowth of bacteria in the intestine as well as altering their relative proportions (Engen et al., 2015). Furthermore, the permeability of the intestine increases, allowing live bacteria and bacteria-derived metabolites such as endotoxins into the circulatory

system. This breach is not just a side effect of drinking alcohol but is known to be causative in the pathology associated with ALD. Research comparing germfree mice (completely sterile and lacking any microbiota) to normal mice reveals that germ-free mice are resistant to the liver damage caused by alcohol. Conversely, if the abnormal microbiota of a human patient suffering from ALD is introduced into germ-free mice, their susceptibility to alcohol-induced liver disease is far higher than that of mice receiving the microbiota of an alcoholic without hepatitis (Llopis et al., 2015). The gut microbiota is thus key to determining susceptibility to ALD as well as to the progression of the disease once it sets in. While the broad mechanisms that link the microbiota to liver disease are known, the host responses that contribute to the disease were not very well understood until now. In this issue of Cell Host & Microbe, Wang et al. (2016) delineate the critical role of intestinal epithelial-cellspecific anti-microbial proteins (AMPs), Reg3g and Reg3b, in ALD. AMPs are secreted by epithelial and Paneth cells in the intestine and are involved in curtailing microbial colonization. These peptides are part of our innate immune defense system; deficiencies in their production and secretion are associated with various diseases such as inflammatory bowel disease. A major class of AMPs produced by intestinal epithelial cells belongs to the C-type lectin protein family that recognizes Gram-positive (Reg3g) and Gram-negative bacteria (Reg3b) (Cash et al., 2006; van Ampting et al., 2012). Reg3g and Reg3b levels are known to correlate with increasing bacterial load in the gut. Surprisingly, despite the bacterial overgrowth usually associated with ALD, the expression of

these two AMPs is specifically downregulated, whereas the expression of other AMPs remains relatively unchanged. In this study, the authors employed knockout and transgenic mouse models to delineate the roles of these two AMPs in the progression of liver inflammation (steatohepatitis). The major hallmarks of ALD are the presence of activated macrophages, production of inflammatory cytokines, and fat deposition in the liver. These are accompanied by intestinal hyperpermeability, as evidenced by higher plasma endotoxin levels. When the authors subjected both wild-type and knockout mice (Reg3g / and Reg3b / ) to chronic alcohol consumption, they observed that the knockout mice displayed heightened disease severity as compared to wildtype mice. All the typical hallmarks of ALD were aggravated in the knockout mice along with an increase in bacterial translocation. The authors were able to detect viable bacteria in both the mesenteric lymph nodes (MLNs) as well as the liver, suggesting that the AMPs have a protective role to play in ALD. Despite the increase in bacterial translocation to other organs, the knockout and wild-type mice were not significantly different in terms of their intestinal permeability after alcohol exposure. In order to elucidate the mechanism behind the increased translocation, the authors characterized the microbiota of both the mice groups. The two groups did not differ in the total number or composition of their luminal bacteria. However, the knockout mice showed a significant rise in the number of bacteria that colonized their mucosa. Under normal conditions, the action of Reg3g and Reg3b restrict bacterial replication in the mucosa. Their absence allows the microbes

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Previews

Figure 1. Ethanol Impairs Host Anti-Microbial Defense to Promote Liver Disease A healthy gut is defined by the balance between its anti-microbial defense and the gut microbiota in the lumen. Levels of the AMPs, Reg3g, and Reg3b are tightly regulated by TLR- and IL-22dependent mechanisms to achieve gut homeostasis. In the presence of optimal levels of these AMPs, the luminal bacteria are unable to colonize the mucosa or cross the tight-junction complexes. Hence, bacteria are unable to translocate to systemic sites such as the liver. In contrast, chronic or binge drinking results in suppression of Reg3g and Reg3b by as-yet-unknown mechanisms. As a result, the mucosa-associated bacterial number increases. This increase, coupled with heightened intestinal permeability, leads to enhanced bacterial translocation to the liver via the MLN and exacerbates ALD.

to come in close contact with the epithelium, thus permitting an increase in bacterial translocation (Vaishnava et al., 2011). Analysis of duodenal biopsy samples from patients with alcohol dependency, versus those from healthy controls, showed a similar correlation between levels of Reg3g expression and mucosal colonization. Interestingly, while the loss of Reg functionality impaired the prognosis of the disease, overexpression of Reg3g in wild-type mice conferred resistance to steatohepatitis; Reg3goverexpressing mice displayed reduced liver inflammation and fat deposition, lowered loads of luminal and mucosa-associated bacteria, and reduced bacterial translocation. The current study is the first to establish a direct link between AMPs and an extra-intestinal disease (Figure 1). It brings forth a unifying principle in the pathology of ALD, where both Reg3g and Reg3b, despite their varying specificities, work together to protect against liver injury. It is not the nature of the bacteria, whether Gram positive or nega-

tive, but rather their ability to translocate across the epithelium that appears to be the determinant of disease progression. Intriguingly, the mechanism underlying bacterial translocation to the liver is still unknown. While intestinal permeability is increased during alcohol treatment, the paracellular spaces are too small to allow live bacteria to pass through. This implies that certain host cells may act as carriers. CD11c+ CX3CR1hi cells are known to be capable of ingesting and trafficking luminal bacteria to the MLN. While the authors found no difference in the migration of these cells to the MLN, alternative mechanisms of bacterial translocation remain to be discovered. Recent research has suggested that the liver can act as a secondary firewall in bacterial defense, and a compromised clearance of bacteria in the MLN may result in live bacteria trafficking to the liver via the portal vein (Balmer et al., 2014). Study of the mechanisms underlying this translocation could reveal novel aspects linking the gut to systemic circulation and immunity.

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An interesting observation made by the authors is regarding the suppression of Reg3g and Reg3b, despite the dysbiosis and bacterial overgrowth observed in ALD. Expression of Reg proteins by epithelial cells is regulated directly in response to bacteria, via TLR-dependent mechanisms, and indirectly via an IL-22dependent pathway (Brandl et al., 2007; Zindl et al., 2013). This raises the question of whether the effect of ethanol on AMP expression is direct or indirect. It is possible that alcohol directly affects innate immune signaling via a TLRdependent pathway in intestinal epithelial cells. Support for this hypothesis comes from studies suggesting that alcohol can interfere with receptor clustering and signaling events downstream of TLRs (Szabo et al., 2007). Alternately, alcohol exposure may result in changes in the level of IL-22 in the gut and interfere with expression of Reg3g and Reg3b. Alcohol might also regulate anti-microbial genes by an epigenetic mechanism. Alcohol is primarily metabolized in the gut and liver, via oxidative pathways, by the action of enzymes such as alcohol dehydrogenase and CYP2E1. These pathways significantly alter the ratio of NADH/NAD+ within cells, thus changing the intracellular redox environment. Such changes can be directly sensed to alter both the epigenetic and transcriptional program of the cell (Zakhari, 2013). CYP2E1-dependent regulation of circadian genes is already known to contribute to tight junction protein suppression and intestinal barrier leakiness upon alcohol exposure. While the effect of diet and metabolites on the microbiota (and hence health) is well established, this study brings forth the fascinating possibility of a direct link between metabolites and regulation of AMPs in the host. Manipulation of AMP levels in the gut might open up new avenues for management of ALD in the future. REFERENCES Balmer, M.L., Slack, E., de Gottardi, A., Lawson, M.A., Hapfelmeier, S., Miele, L., Grieco, A., Van Vlierberghe, H., Fahrner, R., Patuto, N., et al. (2014). Sci. Transl. Med. 6, 237ra66. Brandl, K., Plitas, G., Schnabl, B., DeMatteo, R.P., and Pamer, E.G. (2007). J. Exp. Med. 204, 1891– 1900. Cash, H.L., Whitham, C.V., Behrendt, C.L., and Hooper, L.V. (2006). Science 313, 1126–1130.

Cell Host & Microbe

Previews Engen, P.A., Green, S.J., Voigt, R.M., Forsyth, C.B., and Keshavarzian, A. (2015). Alcohol Res. 37, 223–236. Llopis, M., Cassard, A.M., Wrzosek, L., Boschat, L., Bruneau, A., Ferrere, G., Puchois, V., Martin, J.C., Lepage, P., Le Roy, T., et al. (2015). Gut. http://dx.doi.org/10.1136/gutjnl-2015-310585. Szabo, G., Dolganiuc, A., Dai, Q., and Pruett, S.B. (2007). J. Immunol. 178, 1243–1249.

Vaishnava, S., Yamamoto, M., Severson, K.M., Ruhn, K.A., Yu, X., Koren, O., Ley, R., Wakeland, E.K., and Hooper, L.V. (2011). Science 334, 255–258.

Wang, L., Fouts, D.E., Sta¨rkel, P., Hartmann, P., Chen, P., Llorente, C., DePew, J., Moncera, K., Ho, S.B., Brenner, D.A., et al. (2016). Cell Host Microbe 19, this issue, 227–239. Zakhari, S. (2013). Alcohol Res. 35, 6–16.

van Ampting, M.T., Loonen, L.M., Schonewille, A.J., Konings, I., Vink, C., Iovanna, J., Chamaillard, M., Dekker, J., van der Meer, R., Wells, J.M., and Bovee-Oudenhoven, I.M. (2012). Infect. Immun. 80, 1115–1120.

Zindl, C.L., Lai, J.F., Lee, Y.K., Maynard, C.L., Harbour, S.N., Ouyang, W., Chaplin, D.D., and Weaver, C.T. (2013). Proc. Natl. Acad. Sci. USA 110, 12768–12773.

Microbes without Borders: Decompartmentalization of the Aging Gut Erin S. Keebaugh1 and William W. Ja1,* 1Department of Metabolism and Aging, The Scripps Research Institute, Jupiter, FL 33458, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chom.2016.01.016

The microbiota supports intestinal homeostasis in developing animals. With increased age, gut maintenance declines and microbes can stray from traditional zones, negatively impacting host health. In this issue of Cell Host & Microbe, Li et al. (2016) detail the mechanisms leading to the decline in intestinal health in aged flies. Flies often feed on rotting fruits teeming with microbes, most of which are tolerated by, or are even beneficial to, the developing host. Early-life studies comparing conventionally raised and axenic (germ-free) Drosophila showed that the presence of commensal bacteria supports normal growth. In some circumstances, consumed microbes provide nutritional support or protect the host by outcompeting pathogenic bacteria (Buchon et al., 2013a; Yamada et al., 2015). Other bacteria are actively controlled by a network of immune pathways within the intestinal epithelia. The immune deficiency (Imd) pathway, which generates antimicrobial peptides, is perhaps the most well-defined line of enteric defense. Imd pathway genes are differentially expressed across the fly intestine, which has at least ten different intestinal compartments with unique sets of molecular and anatomical features (Figure 1; Buchon et al., 2013b; Marianes and Spradling, 2013). The Imd pathway is activated by pattern recognition receptors that recognize a peptidoglycan motif possessed by both pathogenic and commensal species common to the fly (Buchon et al., 2013a). Within the intestine,

Imd-negative regulators attenuate the response to less-invasive commensal species, whereas Imd induction is stronger in response to invasive, pathogenic bacteria, which also generate higher levels of immune elicitors. The different levels of immune activation induced by commensal and pathogenic bacteria aid in the appropriate regulation of enteric microbes. With age, however, flies experience a loss of intestinal homeostasis; two hallmarks of aging include alterations in the composition and quantity of gut commensal populations and a decline in barrier function (Clark et al., 2015; Guo et al., 2014). At the molecular level, a decrease in activity of Imd-negative regulators ultimately leads to intestinal stem cell hyperproliferation, failure of barrier function, and organismal death. Blocking Imd deregulation, or raising flies axenically, can prevent some of these effects and extend life, suggesting that agerelated dysbiosis is a major factor in mortality (Clark et al., 2015; Guo et al., 2014). In this issue of Cell Host & Microbe, Li et al. (2016) explore the onset of intestinal pathologies in aging flies, and show that intestinal compartmentalization and the proper maintenance of enteric microbiota

are closely aligned. To determine if intestinal partitions are involved in controlling luminal constituents, Li and colleagues first disrupted compartmentalization in the fly intestine, focusing on the ‘‘copper cells,’’ which form an acidic region that shares similarities to our stomach. When copper cells are intact, microbes are mostly associated with anterior portions of the fly gut (Figure 1). When copper cells are ablated, the discrete acidic region of the gut is lost and commensal numbers increase throughout the intestine, with the largest increase seen in the posterior gut. This suggests that the copper cells—and their corresponding acidic region—are normally responsible for partitioning microbes within the intestinal lumen and lowering commensal counts in posterior intestines. Furthermore, Li et al. (2016) found that disruption of copper cells drove Imd activation and epithelial proliferation, demonstrating a crucial link between compartmentalization and commensal homeostasis, and highlighting the possibility that decompartmentalization may be a primary factor driving intestinal abnormalities in aged animals. Consistent with this idea, knocking down transcription

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Alcohol Lowers Your (Intestinal) Inhibitions.

Alcohol causes microbiota dysbiosis and breaches intestinal integrity, resulting in liver inflammation and ultimately cirrhosis. In this issue of Cell...
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