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Summary: A new study sheds light on how neurons in the motor cortex communicate with muscles and drive motion. Using optogenetics, the researchers discovered the motor cortex communicates with muscles differently, depending on the type of movement an animal makes.
Source: Zuckerman Institute.
Study in mice answers long-standing scientific question about the brain’s ability to drive movement.
New research by Columbia scientists offers fresh insight into how the brain tells the body to move, from simple behaviors like walking, to trained movements that may take years to master. The discovery in mice advances knowledge of how cells in the motor cortex — the brain’s movement center — communicate with muscles, and may help researchers better understand what happens in injury or disease, when the mechanisms that underlie movement go awry.
These findings were reported today in Neuron.
“All movements, from the most basic, like walking, to the most skilled, like playing the piano, require an extraordinary choreography between the brain and the body — a process that we still do not fully understand,” said Thomas M. Jessell, PhD, codirector of Columbia’s Mortimer B. Zuckerman Mind Brain Behavior Institute and the paper’s senior author. “In this study, we were able to view the interaction between brain and body in real time, allowing us to see exactly when the brain’s motor cortex directs muscle movement, and also how that influence actually works.”
Previous studies have shown that the motor cortex, despite its name, is not necessarily required for all types of movement. If an animal sustains damage to the motor cortex, it can recover and walk normally. But more specialized movements, such as precise grasping, do require the motor cortex, and without it, an animal cannot recover.
“We wanted to see how the motor cortex operates during two completely different behaviors — grasping, which appears to require the motor cortex, and walking, which does not,” said Andrew Miri, PhD, a postdoctoral researcher in the Jessell Lab at Columbia University Medical Center (CUMC) and the paper’s first author. “Using optogenetics, a technique which turns cells on and off with light, we silenced the motor cortex in the brains of mice as they either walked on a treadmill or reached to grab a joystick. We could then observe any changes to the animals’ movement in real time.”
After silencing the motor cortex, the researchers noted that it took 10 milliseconds before the animals’ ability to grasp was disturbed. But it took at least 35 milliseconds before they observed any change to the animal’s ability to walk. These findings were a critical piece of the puzzle, because they implied that the animals’ motor cortex is communicating with muscles differently, depending on which movement the animal is making. The question then was: How is this happening?
To find out, the researchers collected electrical recordings from hundreds of individual neurons in the motor cortex while the mice performed the two movements: walking and reaching to grasp. The research team worked with Columbia’s Center for Theoretical Neuroscience to mathematically visualize and quantify was happening in the motor cortex.
“Each neuron emitted a series of impulses during both types of movement. But what was most striking was how the impulses in one pair of neurons could be remarkably in sync during the reach task, while the impulses in those same two neurons were completely mismatched while the animal walked,” said Dr. Miri.
In other words, what mattered was not how one neuron pulsed, but how that neuron pulsed similarly to those around it. The aggregate of such similarities — and differences — across the entire motor cortex, which the mathematical analyses revealed, was the main driver of one type of movement over the other.
“These findings offer for the first time a comprehensive explanation for how the brain’s motor cortex directs only some types of movements, even when always appearing to be active, and may offer clues as to why some movements can be relearned after motor cortex damage while others cannot,” said Dr. Jessell, who is also the Claire Tow Professor of Motor Neuron Disorders in Neuroscience and of Biochemistry and Molecular Biophysics at CUMC.
This study therefore has implications for medicine, said Dr. Jessell.
“Understanding activity in the motor cortex is critical to developing treatments for a range of diseases and injuries of movement,” he explained. “Whether it’s building brain-machine interfaces that can accurately mimic communication between the brain and muscles, or developing a way to diagnose the early signs of movement disorders such as amyotrophic lateral (ALS), today’s findings bring us closer to the detailed understanding of the brain that we need.”
Data Source: 2014 Medicare
Chronic Conditions Data
Warehouse (CCW), Centers for
Medicare & Medicaid Services.
Source: Endocrinology news
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Although genes contribute to colorectal cancer, the gut microbiota are an important player. Accumulating evidence suggests that chronic infection and the ensuing inflammation contributes to tumor initiation and tumor progression. A variety of bacterial species and tumor-promoting virulence mechanisms have been investigated. Significant advances have been made in understanding the composition and functional capabilities of the gut microbiota and its roles in cancer. In the current review, we discuss the novel roles of microbiota in the progression of colon cancer. Although microbiota technically include organisms other than bacteria e.g., viruses and fungi, this review will primarily focus on bacteria. We summarize epidemiological studies of human microbiome and colon cancer. We discuss the progress in the scientific understanding of the interplay between the gut microbiota, barrier function, and host responses in experimental models. Further, we discuss the potential application in prevention, diagnosis, and therapy of colon cancer by targeting microbiota. We discuss the challenges lie ahead and the future direction in studying gut microbiome in colon cancer to close the gap between the basic sciences and clinical application.
Colorectal cancer is the 3rd most common cancer in both males and females in the US and the 2nd leading cause of cancer deaths with the estimated new cases of nearly 133,000 and deaths of 50,000 in 2015.1 Worldwide, 1,360,000 new cases and 694,000 deaths per year are estimated.2 Cancer incidence in the large intestine is also known to be approximately 12-fold higher than that of the small intestine, which has been attributed to several magnitude greater bacterial density in the large intestine (∼1012 cells per ml) compared with that in the small intestine (∼102 cells per ml).3 With advance in metagenomic technology, growing evidence now suggests that dysbiosis, i.e., imbalance in of normal intestinal microbiota, can promote chronic inflammatory conditions and the production of carcinogenic metabolites, leading to neoplasia.4,5
Gut microbiota represents a complex ecosystem that develops in close parallel with hosts and depends on the physiological environment of hosts. Humans have coevolved with their microbes over thousands of years. The gut bacterial population stabilizes during the first years of life and then remains stable throughout our life in terms of the major populations. Human gut microbiota are dominated by four main phyla: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. The corporate number of microbial species in human gut is estimated to be 1000–1150, with each individual harboring at least 160 (Qin, Li et al 2010). The number of genes of gut microbiota exceeds the number of genes in the human genome by 150 times. A large portion (38%) of the total gene pool is commonly shared from individual to individual. The “core human microbiome” refers to the central part of microbial gene pool existing in all or most of humans. The “variable human microbiome” is the microbial genes in a specific cohort of people, which is determined by a combination of host factors (Turnbaugh, Ley et al 2007). In the modern society, the host-microbial relationship is now being dramatically affected by shifts in the collective human microbiome resulting from changes in the environment and societal norms (Sun and Chang 2014).
In this review, we will discuss the roles of gut microbiota in colorectal cancer, summarizing both epidemiologic observations and the data from experimental animals. Although microbiota technically include organisms other than bacteria e.g., viruses and fungi, this review will primarily focus on bacteria, of which significant recent progresses have been made in understanding their role in human health. Specifically, understanding of the interplay between the gut microbiota, barrier function, and inflammatory responses will uncover new therapeutic targets in colorectal cancer. We will discuss the potential application in prevention, diagnosis, and therapy of colorectal cancer by targeting gut microbiota. Moreover, we will also discuss challenges lie ahead and the future direction in studying gut microbiome in cancer to close the gap between the basic sciences and clinical application.
At least two approaches have been employed to study colorectal cancer-associated microbiome. One is the targeted, more hypothesis-testing studies to examine whether exposure to specific bacteria species of interest increases the risk of colorectal cancer. The second type is studies aiming to identify differences in overall microbial composition by disease status. The latter has gained more popularity recently with advances in genomic technology for high throughput sequencing and discussed here first.
Most common materials used in these types of investigation are fecal or mucosal biopsy/resection samples and have been analyzed primarily by pyrosequencing. But it is now clear that bacterial populations in feces and mucosa are distinct.6,7 As summarized in Table 1, the majority of these studies have demonstrated beta diversity by principal coordinate or component analysis illustrating structural difference of gut microbiome, where samples belonging to different disease status (cancer, adenoma, or controls/normal adjacent tissue) cluster in different two dimensional spaces,7–12 indicating the presence dysbiosis. Analysis of community diversity/richness indies based on 16SRNA gene sequencing has shown significantly reduced microbial diversity in feces of colorectal cancer patients than in controls13 and in cancer tissue compared with mucosa at least 10 cm apart from cancer.14 On the contrary a richness index was higher in rectal mucosa of colorectal cancer patients than in that of control subjects7 or in cancer tissues than paired normal tissue.11 Others did not find differences in these alpha diversity indices.9,10,15,16 With or without using additional quantitative PCR (qPCR), these studies have also found that specific bacterial groups were more common or less common in colorectal cancer cases than control specimens.7–16 Because each study has used different taxonomic levels/classifications for the comparison, there have little consistency in changes associated with colorectal cancer. However, there were multiple studies reporting overrepresentation of Fusobacterium and Porphyromonasand and underrepresentation of Faecalibacterium (Table 1). Yet, it should be noted that some of these studies were based on very small numbers of samples and control subjects were often not comparable with cases in terms of basic demographic factors (such as age). In summary, while these studies underscore marked differences in gut microbial membership between colorectal cancer patients and healthy controls, it is difficult to generalize characteristics of cancer associated gut microbiome.
Table 1. Summary of 16rRNA pyrosequencing studies involving colorectal cancer (CRC) and control specimens addressing microbial community structure.
| Authors (year) | Study subjects (N) | Type of specimens | 16S rRNA region | Beta diversity | Alpha diversity | Overrepresentation | Underrepresentation |
|---|---|---|---|---|---|---|---|
| Sobhani et al (2011)8 | CRC (60), colonoscopy control (119) | Stool | V3–V4 | PCA | – | Bacteroides/Prevotella | |
| Ahn et al (2013)13 | CRC (47), surgical control (94) | Stool | V3–V4 | – | Shannon index down in CRC | Fusobacterium, Porphyromonas | Clostridia |
| Wang et al (2012)9 | CRC (46), healthy volunteers (56) | Stool | V3 | PCA | No difference in diversity and evenness | Porphyromonas, Escherichia/Shigella, Enterococcus, Streptococcus, Peptostreptococcus | Bacteroides, Roseburia, Alistipes, Eubacterium, Parasutterella |
| Wu et al (2013)10,65 | CRC (19), healthy volunteers (20) | Stool | V3 | PCoA | No difference in diversity and richness | Bacteroides species Fusobacterium Campylobacter species | Faecalibacterium, Roseburia |
| Weir et al (2013)15 | CRC (11), healthy volunteers (10) | Stool | V4 | – | No difference in diversity and richness | Akkermansia muciniphila | Bacteroides, Prevotella, Ruminococcus |
| Chen et al (2012)14 | CRC (46), healthy volunteers (56) | Stool, rectal swab, cancer tissue, adjacent (2–5 cm and 10–20 cm apart) normal mucosa | V1–V3 | – | Shannon index down in CRC tissue vs paired mucosa 10–20 cm apart | Lactobacillales(tumor), Erysipelotrichaceae, Prevotellaceae, Coriobacteriaceae(stool) | Faecalibacterium(tumor) |
| Mira-Pascual et al (2015)7 | CRC (7), adenoma (11), healthy volunteer (10) | Tissue (tumor or rectal mucosa), stool | V1–V3 | PCoA (tissue) | Richness up in cancer tissue | Enterobacteriaceae(cancer tissue) | |
| Geng et al (2013)11 | CRC (8) | Paired tissue (cancer, normal) | V1–V2 | PCoA | Richness up in cancer | Roseburia | Microbacterium, Anoxybacillus |
| Geng et al (2014)12 | CRC (8), adenoma (10), healthy volunteer (10) | Normal and tumor tissue | V1–V2 | PLS-DA | – | Streptococcus, Porphyromonas, Veillonella (cancer vs control) | |
| Kostic et al (2012)16 | CRC (95) | Paired tissue (cancer, normal) | V3–V5 | – | No difference in richness | Fusobacterium | Bacteroides, Clostridia, Faecalibacterium |
PCA: Principal component analysis; PCoA: Principal coordinate analysis; PLS-DA; Partial least square discriminant analysis.
Streptococcus bovis (SB) is a gram-positive bacterium and lower-grade opportunistic pathogen that can cause systemic infections (endocarditis or bacteremia) in humans. It is a group D streptococcus with the specific ability to grow in 40 percent bile.17 Intestinal mucosal lesions have been deemed to serve as a portal for these bacteria to the systemic circulation. Based on biochemical traits, DNA homology and divergence in 16S rRNA sequences, SB can be grouped into Streptococcus gallolyticus (SB biotype I and II/2) and Streptococcus infantarius (biotype II/1). Earlier studies suggest stronger association of S. gallolyticus with colorectal tumors18 in contract to stronger link of S. infantarius to non-colonic cancers, primarily in the pancreas and biliary tract.19
Although SB is a member of normal gastrointestinal flora in ruminants, e.g., cattle, sheep, horses, pigs, camels and deers, it is also found in human feces as well as gastric biopsy materials.20,21 Approximately 10% of healthy individuals have been estimated to carry this bacterium asymptomatically in their digestive tract.20 While fecal–oral or oral–oral is a possible transmission route between humans, it may be acquired through dietary intake of ruminant-derived foods, such as unpasteurized dairy products,22 red meat and animal organs.20 In fact SB is a frequently detected contaminant in commercially available meat.23,24The correlation between SB and colonic disease has long been recognized. Besides case-reports for the patients who were diagnosed with asymptomatic colorectal neoplasia simultaneously with SB endocarditis or bacteremia,25–30investigators have reported increased prevalence of colorectal tumors (cancer and polyps) among patients diagnosed with SB endocarditis or bacteremia. The prevalence of colorectal tumors ranges from 10 to 60%,18,31–45 although these are based on diverse study populations in terms of patient demographics and colorectal surveillance methods. These variations may also be due to the heterogeneous definition of the cases, as adenomas have been defined as diseased in some studies but not in the others.46 A more recent study found that 52% of SB bacteremia patients had advanced adenoma/cancer, which was approximately 2.5 fold more frequent than colonoscopy controls.47 Similar prevalence (60%) of advanced adenoma/cancer was reported in SB endocarditis patients by Sharara et al.48
The second set of evidence is derived from studies comparing SB prevalence among various patient groups with or without colonic diseases.49–56 While 3 small studies including 13–46 controls and corresponding 11 colorectal cancer, 47 pediatric inflammatory bowel disease (IBD) and 56 polyp patients failed to show any association,52–54 five other studies found that SB carriage (either in stool or antibodies) rates were significantly higher in cancer patients than in controls. Interestingly, 3 studies also showed that patients with premalignant lesions (IBD or polyps) had intermediate SB carriage rate between cancer cases and controls. In addition, stronger associations observed in studies by Darjee & Gibb, Tjalsma et al and Abdulamir et al51,55,56 suggest that antibody assays may be a more powerful tool than fecal culture in assessing the associations between this bacterial infection and colorectal disease. Subsequent enzyme-linked immunosorbent assay (ELISA) based studies have demonstrated that seropositivity or higher antibody titer to specific SB antigens or their combinations was associated with early stage of colorectal cancer57,58or colorectal cancer diagnosed at younger age (<65 years),59 yielding odds ratios of 1.5–8.0. In summary, despite these observations it remains elusive whether colorectal neoplastic sites provide a specific niche for SB resulting in sustained colonization and survival or whether SB infection itself promotes colorectal carcinogenesis, or a combination of both.
H. pylori was designated as a group 1 human carcinogen by International Agency for Research on Cancer (IARC) in 1994 because an expert panel concluded that there was sufficient evidence in humans for the carcinogenicity of this bacterial infection and that its chronic infection causes non-cardia gastric adenocarcinoma and low-grade B-cell mucosa-associated lymphoid tissue lymphoma.60H. pylori is a gastric pathogen that infect more than a half of the adult population in the world.61 Gastric carcinogenic pathway caused by H. pylori has been well documented, arising from stages of premalignant lesions, i.e., chronic gastritis, atrophic gastritis, intestinal metaplasia and dysplasia, and then progressing to adenocarcinoma.62–64 While the gold standard for diagnosis of active Helicobacter infection is histological detection in gastric biopsies, stool antigen tests have been clinically accepted as a non-invasive alternative, indicating H. pylori also resides in the large intestine. Although no Helicobacter induced intestinal pathologies have been established, a number of epidemiologic studies have been conducted to examine if HP infection increases the risk of colorectal cancer. A recent meta-analysis by Wu et al65summarized the data for 3488 colorectal cancer cases, 3792 colorectal adenoma and 10,598 and 4348 corresponding controls from 27 studies. They reported significantly increased summary odds ratios for both, 1.39 (1.18–1.64) for cancer and 1.66 (1.39–1.97) for adenoma. However, two prospective studies66,67 with the nested case–control design did not find any indication of increased risk, while all others were either cross-sectional or retrospective case–control studies. It is noteworthy that except one study by Jones et al,68there was no histological confirmation of presence of H. pylori in colorectal mucosa as others used gastric histology, serology or breath test to assess H. pylori infection. Nevertheless, significantly increased risks of cancer and polyps were observed by Jones et al,68 as well as in an additional study among children with hamartomous (juvenile) colorectal polyps,69 respectively. Despite relatively consistent epidemiologic observations to date, there seems insufficient evidence to support causality of the events. Certain biases may be involved, such as publication bias as reported65 as well as surveillance bias particularly for adenoma. In addition, there may be indirect consequences from gastric pathology, such as hypergastrinemia, which is common in patients with Helicobacter infection and has been hypothesized to stimulate colorectal tumor growth.70
Escherichia (E) coli strains are aero-anaerobic Gram-negative bacteria in the normal intestinal flora. As a commensal, E. coli coexist harmoniously with their mammalian host, promote normal intestinal homeostasis and rarely cause disease. However, some virulent E. coli that have acquired pathogenicity islands can colonize the human gastrointestinal tract and induce disease.71 Mucosa-associated E. coli have been identified more frequently in colon tissue from patients with adenocarcinomas than in controls.72–74 Some E. coli strains harbor a ∼54-kb polyketide synthases (pks) pathogenicity island that encodes multi-enzymatic machinery for synthesizing a peptide-polyketide hybrid genotoxin named Colibactin.75 Carriage of E. coli positive to the pks island or genes in the island has been recently found more common in the mucosa of colorectal cancer and IBD patients than that of control subjects.71,75,76Epithelial proliferation and E. coli colonization density were significantly correlated in the mucosa distant from cancer76 and psk positive cancer specimens showed higher levels of DNA damage than its negative counterparts,77 supporting potential causal link.
The anaerobe B. fragilis is a colonic symbiote with an affinity for mucosal colonization that comprises a relatively small proportion of fecal microbiota (approximately 0.5%–1%). There are 2 molecular subtypes, nontoxigenic B. fragilis (NTBF) and enterotoxigenic B. fragilis (ETBF) and ETBF is now established as a cause of diarrheal disease.78 ETBF pathogenicity is due to the B. fragilis toxin (BFT), a 20 kDa zinc-dependent metalloprotease toxin with 3 isotypes (BFT-1, BFT-2, and BFT-3) and the bft gene is unique, only identified in B. fragilis.78 BFT binds to a specific colonic epithelial receptor activating Wnt and NF-κB signaling pathways with increased cell proliferation, epithelial release of proinflammatory mediators, and induction of DNA damage78,79 and ETBF promotes tumor formation in experimental animals.79,80 Despite these laboratory data, to date only limited data in humans support an association of ETBF with colorectal cancer. Ulger Toprak et al81 reported that 38% of fecal samples from cancer patients were positive to btf gene while only 12% of those from control patients were positive (P = 0.009). Boleij et al82 recently revealed more frequent detection (∼75%) of btf genes in colonoscopic biopsies, particularly among patients with no antibiotic pretreatment and the prevalence was significantly higher in cancer than controls.
F. nucleatum is a Gram negative, non-spore forming, obligate anaerobic of the Fusobacteriaceae family, which consists of 9 genera, including Fusobacteriumand Leptotrichia. Fusobacterium genus includes at least 14 species, several of which (including F. nucleatum) are known pathogens.83F. nucleatum is perhaps best appreciated for its role as a component of oral plaque, where by virtue of its adhesive abilities it serves as a bridge organism between early and late colonizers of this biofilm and consequently is implicated in various forms of periodontal diseases.84
Until recently, F. nucleatum was thought to primarily be a component of the oral microbiota of humans and only an occasional resident of the gut. However, this premise was built on culture-based examination of stool, which usually does not contain high numbers of live, epithelium-associated bacteria. Using metagenomic approaches recently, growing number of studies have reported an over-representation of sequences from F. nucleatum16,85,86 or genus F5,87,88 in tumors relative to control specimens. Two of these by Castellarin86 and by Warren87 were based on RNA, representing transcribing bacteria. These observations were further confirmed by quantitative (q) PCR and in situ hybridization in tumor tissue.16,86,89 Using qPCR, McCoy et al studied F counts in normal rectal mucosa of the cases with or without colorectal adenoma, revealing a 3 fold increase in risk of adenoma among subjects with highest tertile of F counts.90 Ito et al91 also demonstrated that F. nucleatum detection in formalin-fixed paraffin-embedded tissue by qPCR progressively increased with malignant grades of the lesions from hyperplastic polyps to colorectal cancer. Several others found higher fecal carriage of genus F13,85,92 or Fusobacteriaceae family10 in colorectal cancer patients than in control subjects, pointing to a potential tool for colorectal cancer screening.
All F. nucleatum strains may not equal in their virulence potential. F. nucleatumis naturally co-aggregative and would likely exist in the human gut microbiota as a feature of a larger microbial grouping. The ability to adhere to other bacterial species could also enable gene transfer and thus some F. nucleatum strains may acquire genes through horizontal transfer leading to increased virulence,93which suggests that the involvement of F. nucleatum in disease may not be just a function of a direct result of its own virulence. Despite these accumulated evidences, however, whether this association is indeed involved in colorectal carcinogenesis, or simply the result of F. nucleatum exploitation of an ecological niche created as a result of the cancer/tumor microenvironment, remains to be tested in further studies.
S. enterica is a Gram-negative, facultative anaerobe and an intracellular pathogen to both humans and animals, posing a major public health concern worldwide. Non-typhoidal Salmonella is a major foodborne pathogen, with an estimated 93.8 million cases and about 155,000 deaths globally per year.94aCommon sources of infection include contaminated food, such as meat, eggs and produce.94b Outcomes of this bacterial infection vary widely, ranging from mild self-limiting gastroenteritis to the severe systemic infection that can be fatal. Some of these acute infections result in a chronic carrier state excreting the bacteria in stool and urine without symptoms, which represents another transmission mechanism of this bacterium to other humans. Salmonellosis has also been implicated in the development of various chronic sequelae, including reactive arthritis, irritable bowel syndrome, IBD95 and even cancer.96
Two studies from Scandinavian countries have found that the probability of new IBD diagnosis significantly (2–3 fold) increases compared with general population following an episode of non-typhoid salmonella infection, particularly within the first 10 years.97,98 Although data directly linking to colorectal cancer are still limited, Salmonella typhi carries status is well recognized to increase the risk of gallbladder cancer. A meta-analysis by Nagaraja et al demonstrated the summary odds ratio of 3–496 regardless of salmonella detection methods. Furthermore, Kato et al99 recently reported that antibody against Salmonella flagellin was higher in colorectal cancer and pre-cancer cases than controls in two distinct populations in US and the Netherlands and that dietary intake is the one of the mediating factors, supporting a possible link of Salmonella to colorectal cancer.
Several other species of bacteria have received research interest because their bacterial metabolites have potential detrimental effects against colorectal mucosa or may exert potentially beneficial or protective effect towards epithelial cells. These include Desulfovibrio, Enterococcus faecalis due to hydrogen sulfide and superoxide respectively,100Faecalibacterium prausnitzii and Bifidobacteria due to butyrate and lactate, respectively.101,102 The presence or density/quantity of these bacteria in feces or mucosa has been primarily studied by qPCR. However, there have been only sporadic studies reporting a significant association with colorectal cancer itself,101,102 while others found higher prevalence or density of these bacteria in IBD than in controls, which was further correlated with disease activity.4,103,104 In addition to F. nucleatum, Porphyromonas gingivalis, another oral pathogen more tightly associated with periodontal disease has been linked to digestive tract cancer in a seroepidemiologic study. However, the study was too small to separate colorectal cancer from other cancers.105 The potential association of this bacterium with colorectal cancer may be further corroborated by several other metagenomic studies that observed the overrepresentation of genus Porphyromonas or Porphyromonadaceae family in colorectal cancer specimens than control specimens.10,13,92 Overall, the information available thus far for these bacteria is insufficient to address their etiological involvement in colorectal cancer.
As discussed above, growing evidence now point to differential gut microbial compositions or differential prevalence of specific bacteria in colorectal cancer patients in comparison with control subjects. However, there are also abundant data supporting the associations between gut microbiota and several established risk factors for colorectal cancer. Thus, one should consider a possibility that observed difference in microbiota mirror at least in part changes associated with such risk factors. The best example is obesity. Obese and lean individuals are known to harbor different types of gut microbiota.106 While low energy diet induces change in microbial compositions increasing gene richness,107 microbiome itself also contributes energy harvest to the host, as demonstrated in mice models where transfer of obese microbiome to lean animals led to an increase in body adiposity in a diet dependent manner.108,109Other dietary risk factors for colorectal cancer include low fiber and high red meat intake.110 Dietary fiber and resistant starch are well known to stimulate gut bacterial fermentation to generate short chain fatty acids (SCFA) as well as lactate and to increase relative abundance of bacterial groups with the relevant metabolic activities.111 Although meat intake itself has been rarely studied, removal of animal products (vegan diet) was recently tested in a few clinical trials, showing changes in the Firmicutes/Bacteroidetes ratio and abundance of bacteria capable of triggering inflammation.112,113 Moreover, as discussed above, meats are one of the suspected sources of acquisition of specific pathogens, e.g., S. Bovis ad Salmonella enterica. There has been relatively sparse information as to the associations between other risk factors, physical activity, smoking and alcohol, and gut microbiome. A study from Ireland found that athletes hard significantly higher microbial diversity than controls.114Alcoholics have been reported to carry greater abundance of Proteobacteria or its family Enterobacteriaceae115,116 than control subjects. Smoking cessation led to changes in gut microbial composition, increasing some Firmicutes and decreasing some Bacteroidetes and Proteobacteria,117,118 while Kato et al demonstrated a positive association between smoking and Desulfovibrioabundance.119 Since these risks factors are postulated to be involved in multiple mechanistic pathways, contribution of microbial changes to colorectal carcinogenesis remains to be determined.
Despite the presence of certain biological mechanisms possibly contributing to colorectal carcinogenesis (discussed in later sections), the causal association cannot be inferred only from the data from retrospective or cross-sectional human studies. Except a few for H. pylori66,67 and P. gingivalis,105 all other studies identified the exposure, i.e., the presence bacteria or their antibodies to bacteria, was assessed at or after diagnosis of the disease. This makes it difficult to establish the temporal sequence of the events, which came first, bacteria or cancer. Moreover, the presence of the organism may no longer necessary once carcinogenic pathways are activated by infection as seen in the case of HP and gastric cancer. Serum antibody assays can capture past and current infection and have played a vital role in establishing infectious etiology of several types of cancer, including H. pylori and hepatitis viruses,120especially with use of prediagnostic blood samples from prospective cohorts. Thus, development of reliable serological assays is likely to greatly advance epidemiologic studies. However, due to the limitation of serology as well as fecal analyses, i.e., an inability to identify the location of colonization for the bacteria that can colonize at diverse anatomical sites, histological detection of bacteria in cancer and surrounding tissues would also be required to reinforce their causal involvement.
A systemic review summarizes the original articles studying microbiota and colorectal cancer until November 2014. It showed that some bacteria are consistently augmented (such as Fusobacteria, Alistipes, Porphyromonadaceae, Coriobacteridae, Staphylococcaceae, Akkermansia spp. and Methanobacteriales), while other are constantly diminished in colorectal cancer (such as Bifidobacterium, Lactobacillus, Ruminococcus, Faecalibacterium spp., Roseburia, and Treponema). It is clear that bacteria metabolites amino acids are increased and butyrate is decreased throughout colonic carcinogenesis.121
Identification of components of the microbiota and elucidation of the molecular mechanisms of their action in inducing pathological changes or exerting beneficial activities could aid in our ability to influence the composition of the microbiota and to find bacterial strains and components (e.g., probiotics and prebiotics) whose administration may aid in disease prevention and treatment.122
To study the microbiome in colon cancer, researchers have developed various Experimental animal models: gnotobiological model, antibiotic treatment, inflammatory model with increased risk of colon cancer, inoculation of specific bacteria or products in genetic engineering mice.
Gnotobiological model is an indispensable tool for studying the consequences of bacterial colonization. Animals (such as zebrafish, mouse, rat, pig) can be maintained in sterile conditions and colonized with defined microbes. The effects of the germ-free state or the effects of colonization on disease initiation and maintenance can be observed in these experimental models for disease initiation and progression. Using this approach, researchers have demonstrated direct involvement of components of the microbiota (including non-cultivable commensal bacteria) in chronic intestinal inflammation, development of colonic neoplasia, and other diseases.
A variety of bacterial species and tumor-promoting virulence mechanisms have been investigated, using mouse models. There involve bacterial metabolic products, Pathogenic bacterial toxins/virulence factors, and Immune reaction/modulation.
Firmicutes and Bacteroidetes predominate the gut microbiota, followed by Proteobacteria and Actinobacteria, with minor contributors including Verrucomicrobia and Fusobacteria.123Bacteroides and Ruminococcus are consistent with enriched intake of animal sources, while a plant-based diet favors Prevotella.124Prevotella to Bacteroides ratio constitutes an important index for clinical diagnosis. Butyrate-producing bacteria, including Clostridiumgroups IV (Faecalibacterium prausnitzii) and XIVa, Roseburia spp., Butyricicoccus, and lactic acid bacteria (LAB), mainly Lactobacillus and Bifidobacterium, are believed to benefit the host through anti-inflammation, anti-tumorigenesis, and pathogen exclusion.125–127 There is also a metabolic interplay between LAB and butyrate-producing bacteria due to the ability of the latter to feed on lactate.128
It is known that gut microbiota could produce an enormous quantity of molecules interacting with the host. The beneficial effects of gut microbiota on the host are mainly mediated by its metabolites. Short-chain fatty acid (SCFA), including acetate, propionate, and butyrate, are the major end-products of gut bacteria fermentation of dietary fiber. SCFAs, particularly butyrate, are the preferred source of energy for colonic epithelial cells. SCFA promotes and maintains colonic epithelial health through maintaining barrier function,129suppressing colonic cancer,130–132 inhibiting intestinal inflammation (Wu et al 2014), modulating immune response,133 regulating DNA methylation for proliferation,132 and diminishing oxidative DNA damage.134
The balance between two phyla (Firmicutes and Bacteroidetes) appears to be critical to regulating disease progression. Some bacterial species have been implicated in the development of colorectal carcinoma. Using culture methods, Moore and Moore observed that the abundance of Bacteroides and bifidobacteria was associated with increased risk of colon polyps, whereas Lactobacillus and Eubacterium aerofaciens were protective.135 An association between the abundance of Fusobacterium, E. coli, hydrogen sulfide (H2S)-, and bile salt-producing bacteria was associated with increased risk of colon cancer.5,136 Cancer is associated with reduced abundances of Clostridium, Roseburia, Eubacteria spp., and other butyrate-producing bacteria in fecal samples of adenoma subjects compared with healthy controls. Zeller et al85reported that a relative abundances of 22 gut microbial species, such as Fusobacterium collectively associated with CRC. This is the first paper based on the whole sequence of bacterial genes, not 16S. It also compared the bacterial markers with the results of the standard Hemoccult FOBT routinely applied for CRC screening and an experimental CRC screening test based on methylation of the wif-1 gene, a Wnt pathway member. The authors believe that there is a potential to use fecal microbiota markers for early-stage detection of colorectal cancer.
Salmonella infection in humans can become chronic which leads to low grade persistent inflammation.137 These chronic infections increase the risk of several gastrointestinal138 diseases, including chronic cholecystitis and gallbladder cancer.139,140 Recently, Kato et al reported that antibody against Salmonellaflagellin was higher in colorectal cancer and pre-cancer cases than controls in two distinct populations in US and the Netherlands and that dietary intake is the one of the mediating factors, suggesting a potential link of Salmonella to colorectal cancer.99
In animal models, Salmonella and its derivatives have been observed invading transformed tissue more efficiently than normal tissue.141,142Salmonella AvrA is a multifunctional protein that influences eukaryotic cell pathways by altering ubiquitination and acetylation of target proteins.143–149
We reported that AvrA acts as a deubiquitinase to stabilize β-catenin. By suppressing β-catenin degradation, AvrA enhances intestinal epithelial proliferation, thus promoting tumorigenesis.150 We reported that AvrA-enhanced tumor multiplicity and tumor progression. Our studies could suggest biomarkers (such as AvrA level in gut) to assess cancer risk in susceptible individuals and infection-related dysregulation of β-catenin signaling in colon cancer. Another novel finding in our study was that the pathogenicity factor altered tumor distribution. Uninfected mice treated with AOM/DSS developed tumors in the distal colon.150
In contrast, in mice infected with AvrA-expressing bacteria, tumors were found more in the proximal colon. AvrA alters the colonic milieu so as to enhance tumorigenesis in the right colon. Compared with the left colon, the cecum has a greater bacterial load and increased bacterial fermentation that we speculate contributes to this rightward shift in tumors. Increasing incidence in right-sided tumors has also been reported in the Western world.
While increased endoscopic screening that probably clears distal colonic lesions more effectively than proximal colonic lesions, based on our studies, this shift might also reflect changes in the microbiome. However, it remains unclear how many human CRC cases can be attributed to bacterial agents, how these exactly interact with the human host or the microbial community in the gut.
Gut microbiota metabolism could be linked with polyp formation, using mice genetic model.151 A diet reduced in carbohydrates resulted in reduced polyp formation in APCMin/+ MSH2−/− mice. Butyrate, a bacterial product, induced aberrant proliferation and transformation of colon epithelial cells. Treatment with either antibiotics or a low-carbohydrate diet reduced cell proliferation as well as the number of tumors in the small intestines and colons. However as mice microbial ecology is different, compared to human, authors did not found Fusobacterium, which was shown to be link to CRC in humans.
A paper from Journal of Experimental Medicine152 reported that antibiotics prevented polyp formation. Most of the tumor-dwelling bacteria belonged to the Clostridiales family and an upregulation of inflammatory molecules near the polyps. FMT from the untreated mice to the once germ-free mice, the previous germ-free mice developed polyps. If transplanted early embryos of the transgenic mice into females of another, cancer-free mouse strain. Inoculated at birth with the bacteria of their surrogate mothers, these transplanted mice did not develop tumors until 25 weeks, whereas the genetically identical controls had tumors by 12 weeks. This showed that small changes in the gut microbiota could have a large influence on tumor growth. This study indicates that the same genetic mutation in different individuals may have a different outcome.152
One environmental factor – a diet low in fiber – may impact the intestinal microbiota in a way that affects host cell physiology, cellular homeostasis, energy regulation, and/or metabolism of xenobiotics. This in turn may lead to chronic inflammation and CRC. Cancer is associated with reduced abundances of some butyrate-producing species. Transplanting feces from mice with CRC into germ-free mice leads to increased tumorigenesis.153
While emerging evidence suggests a link between the gut microbiota and colon cancer, it is hard to say that certain bacteria strain(s) play a causal role in CRC. Evidence is still needed to determine whether those bacteria enhance the development of the disease or might even play a causal role.
Cancer is fueled by deregulation of signaling pathways in control of cellular growth and proliferation. These pathways are also targeted by infectious pathogens en route to establishing infection. It is established that a single infectious agent, namely H. pylori, hepatitis B virus, plays a causal role in human gastric and hepatic cancers, respectively. The exact roles and mechanisms of microbes on the development of colon cancer in are still unknown and of great interests.
Although genes contribute to colorectal cancer (CRC), the gut microbiota are an important player. Accumulating evidence suggests that chronic infection and the ensuing inflammation contributes to tumor initiation and tumor progression.137,154 A variety of bacterial species and tumor-promoting virulence mechanisms have been investigated, using mouse models. A recent study in mice showed that adenomas cause barrier defects in the colonic epithelium allowing microbial products to drive IL-23/IL-17-mediated tumor growth.155Another study demonstrated that a human colonic commensal bacterium promoted tumorigenesis via activation of T helper type 17 T cell responses.80
Colitis was shown to promote tumorigenesis by altering microbial composition and inducing the expansion of microorganisms with genotoxic capabilities.75Arthur et al reported the intestinal microbiota as a target of inflammation that affects the progression of CRC. Monocolonization with the commensal E. coliNC101 promoted invasive carcinoma in azoxymethane (AOM)–treated Il10−/− mice. Specifically, deletion of the polypetide synthase genotoxin from E. coliNC101 decreased tumor load and tumor invasion in AOM treated IL10 knockout mice. E. coli NC101 mutant without the polyketide synthase (pks) genotoxic island decreased tumor multiplicity and invasion in AOM/Il10−/− mice. Mucosa-associated pks+ E. coli were found in a significantly high percentage of inflammatory bowel disease and CRC patients. These studies have highlighted the essential roles of bacteria and/or their products in colonic tumorigenesis.
SCFA is known to modulate immune responses in intestine.133 Another bacterial product Peptidoglycan (PTGN) modulates peripheral immune function via a pattern-recognition receptor, oligomerization domain-containing protein-1 (NOD1) and depletion of the microbiota in mice.133 Lower systemic PTGN concentration leads to less ability to kill certain bacterial pathogens. Polysaccharide A, produced by a commensal bacteria, increases local interleukin 10 by inducing Foxp3+ regulatory T-cell and this effect is mediated by Toll like receptor 2 signaling.156,157 Although recent studies provide insights into the roles of the bacterial products, the molecular mechanisms of the beneficial effects are not fully elucidated yet.
Analysis of the functions that significantly differed between healthy participants and cancer patients revealed a global metabolic shift from predominant utilization of dietary fiber in the tumor-free colon to more host-derived energy sources in CRC.85 They hypothesize that an increased degradation of host glycans might be related to the etiology of CRC. In healthy gut metagenomes, exclusively some fiber-degrading enzymes and fiber-binding domains are enriched, whereas in CRC metagenomes, the microbiota appeares to exploit growth substrates derived from host cells to a much larger extent.85
In summary, the general mechanisms for bacteria – associated (or induced) GI tumorigenesis are through enhancing toxic bacterial products, decreasing beneficial bacterial metabolites, disrupted tissue barriers. Abnormal immunity, chronic inflammation, and hyperpreliferation also contribute to the progression of cancer (Fig. 1). Microbial pathogens and intestinal inflammation can compromise intestinal barrier function and result in increased gut permeability, translocation of various microbial substances, and immune activation.158aDysbiosis further enhances barrier failure and inflammation. The host factor, such as genetic defect, could enhance the dysbiosis along with the environment trigger and change of dietary (Fig.1). One unanswered question is how microbes affect the intestinal epithelium: Do the bacteria make it more permeable or just capitalize on its pre-existing weak spots?
Fig. 1. Working models of general mechanisms for bacteria – associated (or induced) colon cancer. Through enhancing toxic bacterial products, decreasing beneficial bacterial metabolites, disrupted tissue barriers, translocation of microbes, dysbiosis leads to abnormal immune activation, chronic inflammation, and hyperpreliferation that contribute to the colorectal cancer. The host factor, such as genetic defect, could enhance the dysbiosis along with the environment trigger and change of dietary.
Based on current understandings of the roles of microbiota in GI cancer, targeting the gut microbiota is a promising avenue in order to prevent cancer or at least stop the increase of cancerous cells. O’Keefe et al158b investigated the role of fat and fiber in this association by conducting 2-week-long food changes in volunteers from both populations: African-Americans received an African-style diet high in fiber and low in fat, while rural Africans received a high-fat, low-fiber ‘Western’ diet. They found the food changes led to remarkable reciprocal changes in mucosal biomarkers of cancer risk. The dietary switch also changed the microbiota and metabolism in ways known to affect cancer risk.158b This study suggests the potential of dietary intervention or use of prebiotics in colorectal cancer prevention.
Insights into microbiome and cancer risk also provide the opportunities to use of fecal microbial detection for mass screening and diagnosis. By comparing the fecal CRC data to those of IBD patients the researchers could confirm that the microbial characteristics found in the feces were really specific to CRC and not just indicative of inflammatory intestinal conditions in general. The use of fecal microbial CRC detection for mass screening will depend on the development of procedures that are more cost-effective than the ones we used for research purposes.85
The idea of using bacteria as a potent cancer fighting therapy traces its roots back to the early nineteenth century, when French researchers first noticed that bacterial infections in people with cancer often led to shrinkage of their tumors. Increasing evidence has demonstrated that targeting microbiome can improve therapy effects of anti-cancer drugs. Wallace et al reported that inhibiting an enzyme beta-glucuronidase produced by gut microbiota can improve cancer therapy by preventing the intestinal metabolism of the anticancer drug irinotecan.159 More studies have also shown that gut microbes make three anticancer therapies most effective.160a Melanoma growth in mice harboring distinct commensal microbiota and observed differences in spontaneous antitumor immunity, which were eliminated upon cohousing or following fecal transfer. Bifidobacterium is identified to be associated with the antitumor effects. Oral administration of Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy (checkpoint blockade).160b This study also indicates the importance of gut microbiota in other cancers beyond the GI cancer. Although different bacterial groups are implicated in enhancing cancer therapy, the same endpoint through different drugs and different bugs further indicate the novel role of gut microbiome in health and diseases.
Cachexia is a multifactorial condition characterized by systemic inflammation and severe wasting of skeletal muscle, with or without wasting of adipose tissue that causes considerable morbidity and mortality in cancer patients. Infections and inflammation can lead to cachexia and wasting of skeletal muscle and fat tissue by as yet poorly understood mechanisms. Gut colonization by a strain of E. coli prevents wasting triggered by infections or physical damage to the intestine.161 During intestinal infection with Salmonella Typhimurium or pneumonic infection with Burkholderia thailandensis, the presence of this E. colidid not alter changes in host metabolism, caloric uptake, or inflammation but instead sustained signaling of the insulin-like growth factor 1/phosphatidylinositol 3-kinase/AKT pathway in skeletal muscle, which is required for prevention of muscle wasting. This effect was dependent on engagement of the NLRC4 inflammasome.161 Therefore, commensal bacteria in gut promote tolerance to diverse diseases.
Compromised gut barrier function because of dysbiosis or intestinal inflammation can lead to translocation of microbial substances and the development of systemic inflammation with potential consequences for patients prone to cachexia. A recent study showed that non digestible oligosaccharides modulate the gut microbiota may constitute a new nutritional strategy to modulate gut microbiota with positive consequences on cancer progression and associated cachexia.162 Research is needed to clarify the role of gut microbiota and systemic inflammation in the cause of cancer cachexia. Efforts to preserve the integrity of the gut epithelial barrier and/or limit intestinal inflammation in cancer patients may help avoid the serious metabolic alterations associated with cachexia. Multimodal treatment strategies that include interventions aimed at maintaining gut barrier function and correcting dysbiosis may be used to in controlling cachexia.
Microbiota-based cancer prevention, diagnosis, and therapy are beginning to emerge as researchers learn to ‘decode’ the meaning of human microbiota composition at different stages in cancer.
Growing evidence suggests that human microbiota play novel roles in the progression of colon cancer. The advance of current experimental models and methods allow us to obtain the scientific understanding of the interplay between the gut microbiota, barrier function, and host responses. These insights will leads to uncover new therapeutic targets in cancer. Despite these gains, many challenges lie ahead that make it difficult to close the gap between the basic sciences and clinical application.
We believe the following steps are needed in order to move the current microbiota research into clinical practice. First, we need focus on gaining mechanistic insights. Microbiota functions will be important to be considered. We already generated huge information from microbiota analyses. Based on the genomic analyses, we need analyze the microbiota of individuals. Second, we need simple and low-cost tools to identify key bacteria in patients with colon cancer. For GI patients who will undergo therapy – surgery, chemotherapy – we should follow-up of these bacteria and try to understand why some of those will have very good response to therapy and some others will not. Last, identification of components of the microbiota and elucidation of the mechanisms of their action in inducing pathological changes or exerting beneficial, disease-protective activities could aid in our ability to influence the composition of the microbiota. Understanding gut microbiota in cancer will open a door for the prevention, diagnosis and therapy.
The intestinal mucus is an efficient system for protecting the epithelium from bacteria by promoting their clearance and separating them from the epithelial cells, thereby inhibiting inflammation and infection. The function of the colon inner mucus layer is especially important as this explains how we can harbor the large number of bacteria in our gut. The major component of this mucus system is the MUC2 mucin which organizes the mucus by its enormously large net-like polymers. Pathogenic microorganisms, in turn, have developed mechanisms for circumventing this well-organized mucus protective system.
The gastrointestinal tract has a large surface exposed to the intestinal content and is as such a major entry point for pathogens. The organism’s defense system against this challenge can be viewed as consisting of several ‘levels’. The first is the stratified mucus layer which together with the glycocalyx of the epithelial cells provides physical protection. This first defense line is the focus of this review. The second is the single layer of epithelial cells that form a continuous cell sheet interconnected with tight junctions. Here, the goblet cells produce the mucus, and the other major cell type, the enterocytes, have key regulatory roles in mastering the interaction with the microbiota. Both these defense lines belong to the innate immune system together with the third level – resident macrophages and dendritic cells of the intestinal stroma. Finally, the adaptive immune system builds a fourth defense line both as master regulator and as an inducible system to remove microbiota that have sidestepped earlier defense lines.
A digestive tract is required for all advanced multicellular organisms to provide the host with both sufficient energy and molecular building blocks. The organization of this into a stable system is a formidable challenge as it must degrade and absorb ingested food at the same time as it must handle the load of ingested microorganisms. As we understand it today, this task is performed using basically the same principle in all animals, but with slightly different molecular solutions. The one found in most species is based on mucus layers built around the mucins [1]. This conclusion is based on the observation that the gel-forming mucins appear already at the level of early metazoans during evolution [2]. An exception is the insects, where the intestine is protected by a chitin mesh that is reinforced by mucin type molecules anchored via their chitin binding domains [3]. Utilizing these two molecular solutions, all organisms have a proximal and distal part of their intestine that is protected with a physical barrier interrupted by a less protected central part that allows absorption of nutrients. Importantly, the use of highly glycosylated molecules which cannot be degraded by the host as the main building block is common to both insects and higher multicellular organisms.
The gastrointestinal tract is covered by mucus, as revealed by detailed studies in rodents and humans [4;5]. The stomach and colon have a two-layered mucus with an inner mucus layer that is 50-200 μm thick and firmly attached to the epithelium (Fig. 1). The outer mucus layer is easily removed and has a less defined outer border. In contrast to colon, the small intestine has only one layer of mucus, which is possible to remove by aspiration. Normal mucus is totally transparent and microscopically invisible as it is made up of more than 98% water. This has made the mucus difficult to study, as it will collapse as soon as it is not well hydrated and lost when the tissue is fixed with formaldehyde. Recently, we learnt that the surface of mucus can be visualized with charcoal and that the mucus gel is relatively well preserved in Carnoy’s fixative [4;5]. Fig. 1B shows a schematic view of the mucus system of the intestine.
So far only the mucus of the mouse colon has been thoroughly studied for its proteome [5;6]. These studies have revealed a number of proteins which are likely to be important constituents for the formation of a stable mucus. However, it is already clear that the gel-forming mucins make up the mucus skeleton and provide most of the properties of the mucus. The major gel-forming mucin of the stomach is called MUC5AC and that of the intestine MUC2. These two mucins are more similar to each other than to the three other members found in the human genome. Although the MUC2 mucin is the most well-studied, most conclusions are probably valid for the MUC5AC mucin as well.
The MUC2 mucin encodes a protein of approximately 5,200 amino acids (an exact figure cannot be provided as the gene has not been fully sequenced yet) [7;8]. Its central part contains two so-called PTS domains [2] which are rich in the amino acids proline (P), threonine (T) and serine (S) [9]. PTS domains are often highly repetitive (as in the second MUC2 domain) but lack sequence conservation between species. The abundant hydroxy amino acids act as attachment sites for the O-glycans. Once the mucin apoprotein reaches the Golgi apparatus, it is densely decorated by consecutive additions of monosaccharides, a modification which turns these domains into long, stiff bottle brush-like rods where the glycans make up to more than 80% of the mass (Fig. 1A). These highly glycosylated domains are called mucin domains and characterize all mucins. The O-glycans make the mucin domains highly protease resistant [10] and give mucins their high water-binding capacity. Commensal bacteria use these glycans as an important energy source [1].
The MUC2 mucin forms an enormous net-like polymer. The C-termini of individual MUC2 molecules are linked by disulfide bonds into dimeric and the N-termini into trimeric complexes (Fig. 1A). Theoretically, this will lead to flat net-like polymers built around 6-cornered rings as shown in Fig. 1A, right [11;12]. This type of structures have the potential to spontaneously form sheets which in turn would give rise to stratified mucus layers. Such stratification has indeed been observed in the inner mucus layer of colon [5]. In addition to the disulfide bond-stabilized core of the MUC2 mucin, there are additional covalent cross-links that are less well studied [6;13;14]. Moreover, the MUC2 mucin, as well as most other gel-forming mucins, contain CysD domains [2]. In MUC2, one such domain is found on each side of the small mucin domain. CysD domains have been shown to engage in non-covalent dimeric interactions and by this they have the potential to stabilize the interaction of different MUC2 sheets in the stratified mucus layer [15]. Together, our current understanding of mucins suggests that most of the properties of mucus is built into the mucin macromolecule and the network formed upon its polymerization [9].
The inner layer of the colonic mucus is attached to the epithelium, shows a compact and stratified appearance and ranges in thickness is from 50 μm (mouse) and up to several hundred micrometers in humans. Its most remarkable feature is that this inner mucus layer normally is devoid of bacteria. The mucus probably accomplishes this by acting as a filter where for example bacteria are too large to enter [5;9;16]. The inner mucus layer is renewed from below by secretion of the goblet cells and at its lumenal border it is converted to the outer mucus layer (red arrows in Fig. 1B). This outer layer is not attached to the epithelium and expands 4-5 fold in volume upon conversion due to endogenous protease activities acting on the MUC2 mucin. Expansion allows the endogenous bacteria to enter the outer mucus layer, creating a habitat for the intestinal microbiota [9].
The human commensal flora is dominated by Fermicutes and Bacteriodetes [17]. There is currently no deeper understanding of why humans have this flora and why other species have other types of bacteria. However, it is interesting to consider that the O-glycans found on the MUC2 mucin of sigmoid colon differ from those of the small intestine, stomach, and lungs by being almost identical between different individuals irrespective of their blood group status [18]. This could suggest that the mucin glycans are part of a mechanism for selecting our microbiota, maybe because the bacteria carries adhesins that are compatible with the host glycans.
The commensal bacteria have a high proportion of their genomes devoted to enzymes involved in glycan degradation [19;20]. These are typically exoglycosidase in nature, removing one monosaccharide at a time [21]. As the number of different glycans on a single MUC2 molecule is high, typically >100 different oligosaccharide species, many different enzymes would be required for their digestion [18]. This also suggests that the removal of all the glycans will take time, something that is probably important for the protection of colon. Dr. Lijun Xia et al. could recently show that mice lacking the Core 1 enzyme (which synthesizes the major type of extension of the GalNAc attached to the protein core) develop spontaneous colitis [22]. Although, the mechanism for this is not yet established, it is likely that the inner mucus layer of the mutant mice is less efficient in excluding bacteria and the mucins are probably degraded quicker. Preliminary estimates of the MUC2 mucin turnover in the distal colon suggest a half-life of only a few hours, implying that a continuous renewal of the inner mucus layer is crucially important.
The released mucin monosaccharides are used by the commensal bacteria as an energy source in addition to undigested carbohydrates from the food [1]. The relative importance of these different carbohydrate sources is not well studied, but would of course vary dependent on the food intake. In any case, the released monosaccharides are converted into short fatty acids by bacterial metabolism and these can diffuse through the inner mucus layer. By this route, the carbohydrates provide a major energy source to the epithelium and the host. As a large amount of energy is spent on building the quickly turned over mucins and mucus, recovery of most of this energy with the help of the commensal bacteria is probably important for the host.
The small intestine is covered by a single layer of mucus built around the MUC2 mucin [4;7;8]. This mucus is not attached to the epithelium and is also more permeable to bacteria (Fig. 1B). The mucins are normally released at the crypt openings and the mucus will cover the villi. The small intestine has a low bacterial density compared to colon, due to the fast transit and its efficient trapping and distal transport of bacteria [23]. Hooper et al have recently shown that the antibacterial protein RegIIIγ secreted by the enterocytes limits the contact of the gram-positive bacteria with the epithelial cells, but does not lower the number of bacteria in the intestinal lumen [24]. In the absence of this protein, bacteria come in contact with the epithelial cells. RegIIIγ is retained in the mucus after its secretion and by this generates a physical separation between the epithelium and bacteria [23]. The antibacterial peptides produced by the Paneth cells keep the crypts free of bacteria and probably also contribute, together with secreted IgA, to further limiting bacterial growth in the small intestine [25].
Bacteria can be bound to the mucus in several ways. First, the bacteria can be trapped in the pores of the MUC2 network although the small intestinal mucus has larger pores than in colon. Secondly, although the mucins are largely hydrophilic due to their glycan decoration, they also have hydrophobic properties probably conferred by their CysD domains [15]. These domains are suggested to be a major reason of the well-known stickiness of mucus. Thirdly, the polymorphic and variable mucin glycans will bind bacteria carrying adhesins that have specificities for these glycan structures.
The major function of the mucus is to limit bacterial contact with the epithelium and transport bacteria distally [26]. However, this system is not perfect and especially pathogenic bacteria have developed methods to circumvent it. As we still lack some understanding of how mucin and mucus function, we only have limited knowledge of the mechanisms for how pathogenic bacteria avoid this primary protection system. A few aspects and examples will be discussed here.
Pathogens infecting the small intestine have to avoid being trapped by the mucus. It is important for these pathogens to move efficiently towards the epithelium and against the flow caused by secretion and renewal of the mucus. This is reflected in the observation that many intestinal pathogenic bacteria have flagella [27;28]. Bacteria lacking adhesins specific for the mucin glycans of the specific host are probably better off in this environment as they will not be as efficiently trapped. Interestingly, it was noticed several years ago that bacterial adhesins often bind to glycosphingolipids which carry several glycan substructures that are not found among the mucin glycans [29;30]. As glycosphingolipids are abundant in the apical membranes of the enterocytes, these could act as anchoring sites for the bacteria once they have reached the epithelial cells. This and other similar mechanisms will allow specific bacteria to reside below the mucus layer.
Salmonella is an invasive bacterium which needs to circumvent the innate immune system to reach the epithelium [27]. In order to come in contact with the epithelium and swim against the mucus flow, its flagella must be able to propel the bacteria towards the epithelial cells directed by chemotaxis [28]. It has also recently been suggested that the IFNγ receptor mediated signaling triggered by Salmonella infection can affect the goblet cells in a for the bacteria beneficial way, something that could further facilitate bacterial penetration [31].
Helicobacter pylori – The stomach has a two layered mucus system similar to the one found in colon (Fig. 1B) [4]. The attached inner mucus layer maintains a pH gradient with a pH of 2 at the lumen and 7 at the epithelial surface [32]. Few bacteria have developed mechanisms to infect this organ, but H. pylori is a specialist which can cause both gastric ulcer and cancer. An important virulence factor is the adhesin BabA which binds the blood group antigen Lewis b (Leb) and related antigens [33]. As mucins of the stomach also carries Leb antigens, modulation of attachment is probably dynamic during the primary infection when H. pylori penetrate the mucus [34]. In fact, recent observations suggest modulation of BabA expression during colonization [35]. It is also possible that H. pylori mucus interactions can be altered by the mucus pH gradient. The inner mucus layer of the stomach can drastically change its properties as it opens pores in the mucus, probably in a pH-dependent manner, to allow the secretion of hydrochloric acid from the glands [36]. It has also been suggested that H. pylori can alter the mucus viscosity while moving through the mucus gel [37]. Lindén et al. could recently show that also the non-gelforming transmembrane MUC1 mucin limits H. pylori infection both by steric hindrance and by acting as a releasable decoy which washes away the bacteria [38]. The mechanism by which H. pylori penetrates the mucus and infects the host is however complex and far from understood [34].
The parasite Entamoeba histolytica has a Gal-binding lectin that anchors the parasite to the colon mucus, probably to the attached inner mucus layer. This allows the parasite to reside in the colon [39]. The specificity for Gal/GalNAc of this lectin is in line with E. histolytica binding to the glycans on the MUC2 mucin [18]. Once bound, the parasite turns on a program which induces the expression of a cysteine protease necessary for invasion [40]. This enzyme is able to degrade the MUC2 mucin by cleavage at a very specific site localized N-terminally of the first Cys amino acid in the MUC2 C-terminus [41]. The cysteines in this part of MUC2 form numerous disulfide bonds which stabilize the protein and make it resistant to many proteases. A second E. histolytica cleavage site further C-terminally is surrounded by disulfide bonds and a cleavage here will not disrupt the polymer. Cleavage with the E. histolytica cysteine protease at the first site will dissolve the inner mucus layer of colon and thus allow the parasite to reach the epithelium. E. histolytica can then attach to the epithelium and penetrate the intestine to cause the systemic infection typical of this disease. The amino acid sequence containing the E. histolytica cleavage site in the human MUC2 polymer is absent in rodents, something that may explain why rodents are not infected with E. histolytica [41]. It should also be noted that only a portion of humans infected with E. histolytica have invasive disease, a finding that might be related to variable MUC2 glycosylation which may affect cleavage at this site.
Simple experiments have shown that fecal bacteria can secrete proteolytic activities that are capable of cleaving the protein core of the MUC2 mucin [42]. This suggests the existence of bacteria that can cleave MUC2 and by this maybe also disrupt the MUC2 network. Interestingly, several commensal bacteria belonging to the Lactobacilli and Bacteroidetes species do not secrete such proteases [42]. This observation suggests, as expected, that commensal bacteria are not able to disrupt the inner mucus of colon. However, there are other bacteria which can dissolve the inner mucus and if these bacteria are efficient, these and other bacteria can reach the epithelial cells. This situation is similar to the one observed in mice lacking the MUC2 mucin or having defects in MUC2 glycosylation, where severe colitis develops [22;43;44].
The robustness of the inner mucus layer in maintaining a bacteria-free shield is challenging for pathogens affecting the colon [26]. Little is known of this, but in a mouse colon infection model (Citrobacter rodentium), the bacteria reside below the inner mucus layer by a mechanism similar to one described for H. pylori [45].
In the absence of MUC2, as in the Muc2-null mice, the bacteria are in direct contact with the epithelial cells. The bacteria penetrate down into the otherwise sterile crypts and are also found inside the enterocytes [5;43]. These mice develop, depending on the animal housing and genetic background, a more or less severe colon inflammation with infiltration of both neutrophils and lymphocytes, diarrhea, rectal prolapses and failure to thrive [5;46]. The Muc2-null mice also develop cancer after several months, further suggesting that the phenotype of these mice resembles the human disease ulcerative colitis [43]. A further illustration of the importance of the inner mucus layer for protection of colon is shown by the observation that the most commonly used model for colitis in rodents, dextran sulfate treatment, causes the inner mucus layer to become permeable to bacteria several days before any inflammation is observed [16].
The importance of the intestinal mucus has recently been rediscovered, something that is timely with the characterization of the human intestinal microbiota by novel sequencing methods. The organization of the inner mucus layer of colon is very important for homeostasis as defects in or loss of this allows bacteria to reach the epithelium and trigger inflammation.
By John Cassidy
July 19, 2017
In blithely threatening to let the Obamacare exchanges collapse, Trump is ignoring his duty as President to protect the welfare of the citizenry.
Illustration by Doug Chayka
Has there ever been a more cynical surrender of Presidential authority? The editorial board of the Washington Post posed this question on Tuesday, after Donald Trump reacted to the collapse of the Senate health-care-reform bill by suggesting, in a tweet, that his fellow-Republicans should now “let Obamacare fail” and then look to build a new system out of the wreckage. Ignoring the turmoil that such a course of action would generate for tens of millions of Americans, Trump restated his support for it later in the day, saying, “I think we’re probably in that position where we’ll let Obamacare fail. We’re not going to own it. I’m not going to own it. I can tell you, the Republicans are not going to own it.”
The Post’s editorial called Trump’s suggestion “irresponsible,” which was too kind. The first duty of any President is to protect the welfare of the citizenry. In blithely threatening to allow the collapse of the Obamacare exchanges, through which some twelve million Americans have purchased health insurance, Trump was ignoring this duty. Arguably, he was violating his oath of office, in which he promised to “faithfully execute the office of the President of the United States.”
As President, Trump has continued to live by the one imperative that has propelled him for decades: furthering his personal interests and defending his bountiful amour propre. When virtues like consistency, practicality, and decency come into conflict with this overriding concern, they are invariably relegated to secondary status, or ignored completely.
This week, however, Trump’s reaction to his party’s political setback wasn’t just morally abysmal; it was also self-defeating. Thursday will mark the end of his first half year in the White House, and Trump’s Presidency is in crisis. He is facing record low approval ratings, his policy agenda is stalled, and every day seems to bring another damaging revelation about his relationship to Russia—the latest being the news that he had a second, undisclosed conversation with Vladimir Putin during the G-20 summit.
As the Wall Street Journal’s editorial page pointed out on Tuesday, Trump “somehow seems to believe that his outsize personality and social-media following make him larger than the Presidency,” but if he doesn’t change his approach the “brutal realities of Washington politics” will “destroy” him. The Journal was referring to Trump’s attitude toward the Russia investigation, but the point also applies to his approach to policy. By championing the agenda of the Republican right and making the repeal of Obamacare the first big project of his Presidency, he has hung a huge anchor around his own neck.
Over the past six months, Obamacare’s approval ratings have steadily increased and support for the Republican alternative has steadily declined. Last week, pollsters working for ABC News and the Washington Post asked Americans whose health-care plan they preferred: half said Obamacare, while just twenty-four per cent said the Republican plan. Even among self-identified Republicans, just six in ten respondents picked the G.O.P. plan.
A more seasoned and more engaged President would have sensed early on that repealing Obamacare was a potential disaster in the making. Now Trump has Republican politicians breaking ranks, such as Senator Shelley Moore Capito, of West Virginia—a state that Trump won handily in November—who raised concerns about reversing the Affordable Care Act’s expansion of Medicaid, which has enabled some fourteen million Americans to get health care. In Republican-run Arkansas, three hundred and thirty thousand people have been added to Medicaid’s rolls under the A.C.A. Speaking on NPR on Wednesday, Asa Hutchinson, the state’s Republican governor, rejected Trump’s call to let Obamacare collapse and argued instead that lawmakers would have to find a bipartisan solution.
In theory, at least, Trump could have anticipated some of this and responded to the collapse of the Senate bill by aligning himself with Capito and other repeal-skeptical Republicans. Rather than lashing out, he could have announced that he was now eager to work with members of both parties to do what he promised during the campaign—provide affordable health care for everybody, reduce drug prices, protect Medicare—and made it clear that, in the interim, he would support efforts to stabilize the Obamacare exchanges. Conservative Republicans would have accused him of betrayal, but so what?
On the campaign trail, Trump did promise to repeal Obamacare, but the pledge wasn’t central to his appeal as a candidate. He ran as an economic nationalist and an ethno-nationalist. His signature issues were protecting American jobs, keeping out undocumented Latino immigrants, and barring Muslim visitors from America’s shores: the underlying theme was a desire to promote the security of white, native-born Americans. Since becoming President, he hasn’t abandoned this illiberal but fairly popular agenda, and yet it has been overshadowed by the congressional effort to replace Obamacare.
The fact is that the Republican establishment’s desire to roll back the welfare state isn’t consistent with Trump’s stated wish to transform the G.O.P. into the the party “of the American worker.” Going all the way back to Bismarck, the most successful nationalist politicians of the right have supplemented nationalism and economic protectionism with a willingness to expand government programs that provide security for the masses in other areas, including health care. Many of Trump’s European counterparts, such as Marine Le Pen, of France’s far-right National Front, and Norbert Hofer, the head of the Freedom Party of Austria, are committed to universal health care, just as long as it is reserved for native citizens.
After the collapse of the Senate health-care bill, Trump could move in this direction—but that would mean breaking with the Republican leadership, many of its Washington-based activists, and the ultra-conservative big donors who bankroll the Party. It would also involve Trump demonstrating, counter to all the evidence, that he is more than a sham populist. Ever since he made it to the White House, he has been content to champion the G.O.P.’s deeply regressive agenda, despite the dire consequences it would have for many people who voted for him. On Wednesday, Trump changed his tune (again), telling Republican senators during a meeting at the White House that they should stay in town until they find a way to replace and repeal Obamacare. But, of course, he has no plan of his own, and no clue.
Trump is what he is: a self-obsessed carnival barker with authoritarian instincts and little grasp of policy or history. In the long run, his unwillingness (or inability) to change strategy means he is unlikely to go down in history as a transformative figure but, rather, one who exploited a unique set of conditions to win the Presidency. In the short run, Trump’s vacuousness means he has nothing to offer except a laughable effort to blame the Democrats for what has happened. “They’re responsible for passing Obamacare,” Trump’s spokeswoman, Sarah Huckabee Sanders, said on Tuesday. “They’re responsible for the mess we’re in.”
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