微生物群与肠道炎症

孙 俊,金大鹏

(拉什大学 医学中心生物化学系，伊利诺伊斯 芝加哥 60612，美国)

Inflammatory bowel diseases (IBD) include Crohn’s disease (CD) and ulcerative colitis (UC). It is a well-accepted theory that the interactions of various factors contribute to chronic intestinal inflammation in a genetically susceptible host. These factors include genetic risks, environmental triggers, immune responses, and gut bacteria[1]. Many recent reports have strongly indicated that gut microbiome is an essential factor driving the inflammatory process in human IBD[1-2]. In this review, we introduced the general functions of gut bacteria, the association of microbiome and IBD, the experimental models to study microbiome, and the clinical applications of microbiota in IBD. We also discussed the potential therapeutic strategies that can be used to manipulate the gut microbiota, recognizing the limits and challenges in the field.

1 The gut flora is an organ in human body

In traditional medical textbooks, the concept of gut flora or microbiome is hardly discussed. The gut flora is a forgotten organ[3]. The contribution of gut microbiome to pathogenesis is largely unknown until recent years due to the significant progress in the microbiome field. The 2005 Nobel prize in Physiology and Medicine awarded to Robin Warren and Barry Marshall is a reminder that the solution to some human diseases does not reside solely within the host but rather might be found at the interface with the microbial environment. As reviewed by O’Hara AMetal, “the intestine is adapted to bi-directional host-flora exchange and harbors a diverse bacterial community that is separated from the internal milieu by only a single layer of epithelial cells”[3]. The gut flora is of functional importance like “an organ within an organ”[3].

Bacterial cells within the intestine (commensal microflora) outnumber the eukaryotic cells of the body by about 10 times[4]. Based on cell number, each of us is 90 percent microbial and 10 percent human. Human gut harbors 1 000 to 1 150 microbial species, with the number being at least 160 in each individual[5]. The genomes of our gut flora probably contain 100 times more genes than our own genome. Interestingly, the total weight of gut bacteria is about three pounds (1.36 kg).

The densities of living bacteria can be as high as 1011or 1012cells/g of luminal contents[6]. These concentrations are similar to those found in colonies growing under optimum conditions over the surface of a laboratory plate[6]. In the stomach and duodenum, the bacteria are 101to 103CFU/mL. In the jejunum, they are 104to 107cfu/ml. The colon has the highest bacterial numbers,i.e., 1012to 1013CFU/mL. It was long believed that the esophagus is sterile. However, recent studies showed that microbiome are associated with the esophagus too. In the human distal esophagus, inflammation and intestinal metaplasia are associated with global alteration of the microbiome. These findings raise the issue of a possible role of dysbiosis in the pathogenesis of reflux-related disorders[7]. It is believed that the majority of gut bacteria are in lumen. A recent study showed that there are crypt-associated bacteria as well[8].

The proximal colon differs from the distal in that it has higher pH value and active bacterial fermentation[6]. Another key factor influencing the microbial activity and competition is the relative availability of carbohydrate energy sources and nitrogen sources. Less fermentable carbohydrate reaches the distal compared to the proximal colon, leading to differences in the amount of digestible carbohydrate relative to endogenous protein along the colon. Protein-rich diets may increase the amount of dietary protein reaching the large intestine. Peptide supply and pH value can also dramatically alter bacterial populations and short-chain fatty acid composition within microbial communities residing in the human colon[9]. Many colonic mucosal genes that are highly regulated by microbial signals are differentially expressed along the rostral-caudal axis. This suggests that differences in regional microbiota may exist.

Routes for initial colonization of intestinal microbiota include birth canal, breast feeding and womb[10]. The bacterial profile differs vastly in vaginally delivered babies from in those delivered by caesarian sections[11]. The gut flora is then shaped by sporadic environmental events, and gradually become globally homeostatic thereafter, if there is no therapeutic or pathogenic intervention[12].

The structure and composition of the gut flora reflect natural selection at both the microbial and host levels, which promote mutual cooperation within and functional stability of this complex ecosystem[3]. Gut flora participates in a wide variety of physiological functions including fermentation, detoxification, immunomodulation, and exclusion of pathogens[13], which are vital, because in the absence of gut microbiota or with its ablation with broad spectrum antibiotics, and significant consequences can be manifested, e.g. improper development of the gut immune system, development ofC.difficileinfection (CDI) and antibiotic-associated colitis.

Taken together, it is reasonable to view microbiome as an organized system of cells dominated by 4 bacterial phyla (Actinobacteria,Bacteroidetes,Firmicutes,Proteobacteria), and an organ with weight >1 kg and functions closely related to both local and systemic health.

2 IBD and gut flora

The imbalance and instability of the gut bacterial community is called “dysbiosis”. IBD onset is a combinatory result of intestinal microbiome and genetic susceptibility, which are not mutually exclusive and arise from both host and environmental factors that can trigger or contribute to the chronicity of disease. The gut microbial community of IBD patients is different from that of the healthy individuals. Although many recent reports have strongly indicated that gut flora are an essential factor that drives the inflammatory process in IBD, but no single organism has been proven a cause of the pathogenesis in IBD. The contribution of commensal bacteria, the candidate pathogenic bacteria, and the effectiveness of anti-infection treatment were extensively discussed in our previous review[14].

3 Experimental models for IBD and microbiome

The traditional methods to study the gut flora include direct microscopic examination and culture in media. Most bacterial species cannot be cultured, but modern molecular methods, such as broad-range sequencing of conservative 16S ribosomal RNA from amplified bacterial nucleic acid extracted from feces or biopsies. The 16S method used to investigate the diversity of human microbial flora was firstly reported in 2005[15], which indicating evolutionary divergence can be used to identify and classify bacteria. The availability of bacterial sequence data has facilitated the development of molecular probes for fluorescenceinsituhybridization (FISH), and DNA microarrays and gene chips make it possible to specifically identify certain species[3]. These molecular approaches have been used to examine the individuality and stability of the flora overtime and to detect shifts in its composition after weaning, or exposure to antibiotics or dietary changes.

Colonizing previous germ-free animals (such as zebrafish, mouse, rat, or pig) with interested bacterial strains (mono-association) is a state-of-the-art method to understand the function of certain bacterial species in host physiology and pathophysiology. The resulting data can provide a plausible mechanistic basis to explain prevalence of IBD in genetically susceptible hosts. In the gnotobiotic facility, food, air, and water are sterile and animals are kept in an isolator. To ensure sterility, these animals must be screened frequently for contamination. The traditional screening approaches are culturing and Gram staining of feces. Using Germ-free animals, we learned the lessons of life without bacteria. Germ-free animals are more susceptible to infection and have reduced vascularity, digestive enzyme activity, muscle wall thickness, cytokine production and serum immunoglobulin levels, smaller Peyer’s patches and fewer intraepithelial lymphocytes, but increased enterochromaffin cells[16]. However, reconstitution of germ-free mice with an intestinal microflora is sufficient to restore the mucosal immune system[17]. In some instances, it has been possible to induce colitis in a susceptible murine strain with a single species of normal bacteria, for example,Bacteroidesvulgatusin the IL10-deficient mouse[18]. For example, it is believed that the composite human microbiome of Western populations has probably changed over the past century, and brought on by new environmental triggers that often have a negative impact on human health. Using the gnotobiotic models, a recent study[19]showed that consumption of a diet high in saturated (milk-derived) fat, but not polyunsaturated (safflower oil) fat, changes the conditions for microbial assemblage and promotes the expansion of a low-abundance and sulphite-reducing pathobiont,Bilophilawadsworthia. This was associated with a pro-inflammatory T helper type 1 (Th1) immune response and increased incidence of colitis in genetically susceptible IL10-/-mice,but not in wild-type mice. This report also showed that dietary fats, by promoting changes in host bile acid composition, can markedly alter conditions for gut microbial assemblage, resulting in dysbiosis (imbalanced bacterial profile) that can perturb immune homeostasis[19]. These studies provide compelling evidence that the nature of the host defenses, rather than the biological properties of a luminal bacterial species per se, may determine the functional outcome of that interaction.

4 Signaling pathways that connect host and bacteria in intestine homeostasis

4.1 TLRs and NOD The family of Toll-like receptors (TLRs) and the nucleotide-binding oligomerization domain/caspase recruitment domain isoforms (NOD/CARD) are two major host pattern recognition receptor (PRR) systems that regulate the innate immune system. In the intestine, PRRs seem to be crucial for bacterial-host communication. TLRs and NOD proteins are expressed by surface enterocytes and dendritic cells[20]. These PRRs have a fundamental role in response to specific microbial-associated molecular patterns. For example, TLR2 is activated by peptidoglycan and lipotechoic acids; TLR4 is by lipopolysaccharide; and TLR5 is by flagellin; and NOD1/CARD4 and NOD2/CARD15 function as intracellular receptors of peptidoglycan subunits.

Signals from microbes that reside the intestine can be recognized by epithelial TLRs. If the immune activation and tolerance are well balanced, the threat of gut flora is virtually defused[21-22]. A study by Rakoff-Nahoum and colleagues (2004) revealed that commensal bacteria play a protective role against intestinal injury by activating TLRs[23]. Surprisingly, this interaction is required to maintain the architectural integrity of the intestinal surface. Thus, it seems that the epithelium and resident immune cells do not simply tolerate commensal bacteria, but are dependent on them. Recent studies showed that Nod2, an IBD risk gene, is associated with the occurrence of dysbiosis[24-25]. These data further demonstrate the critical role of host-bacterial interactions in intestinal robustness.

4.2 NF-κB Nuclear factor-κB (NF-κB) is a family of transcription factors that plays an essential role in innate and adaptive immune responses. NF-κB is active in the nucleus, and its activity is inhibited by the inhibitor of κBα (IκB). NF-κB is one of the most important regulators of pro-inflammatory gene expression, which regulates the synthesis of quite a few cytokines, including TNF-α, IL-1β, IL-6, IL-8,etc. It is known that NF-κB is highly activated in IBD patients. The signaling of TLR and NOD also regulate the NF-kB activity. Therefore, NF-κB-targeted therapeutics might be effective in IBD. For example, some of the effects of corticosteroids, used in the treatment of IBD are probably mediated through the inhibition of NF-κB activation. Sulfasalazine and leflunomide block nuclear translocation of NF-κB through inhibition of IκBα degradation. This might be caused by a direct effect on IκB kinase (IKB kinase) or upstream signals. Aspirin appears to function as a competitive inhibitor of IKK-β. Agents that block proximal cytokines, such as IL-1 and TNF-α, also limit NF-κB activation and inhibit the inflammatory cascade. Probiotics may provide clinical benefits by ameliorating colitis through inhibiting the NF-kB activity[26].

4.3 Vitamin D/vitamin D receptor Vitamin D levels and vitamin D receptor (VDR) expression are inversely related to human IBD and experimental colitis. Vitamin D deficiency may contribute to IBD as an environment factor. At higher latitudes, cutaneous vitamin D3synthesis is insufficient with lower solar ultraviolet B in winter, which, without vitamin D rich diets, leads to seasonal variations in circulating vitamin D3levels and widespread vitamin D deficiency[27-28]. Prevalence of IBD is higher in the northernmost parts of Europe and America[29]. Patients with IBD have lower serum vitamin D3levels than healthy controls[30]. The proportion of vitamin D deficiency in Australian children with IBD was higher than that in healthy controls[31]. VDR expression at mRNA and protein levels is significantly decreased in IBD patients[32-33]. In mouse models, VDR expression is required to control inflammation. VDR-/-mice were more susceptible in DSS-induced colitis[34-35]. A downregulation of VDR promotes the severity, extent, and duration of mucosal inflammation.

The target genes of VDR signal include the enzyme Cyp24 and antimicrobial peptides (AMPs)[36]. Vitamin D/VDR is responsible for intestinal homeostasis and host protection from bacterial invasion and infection. When the immune system is challenged by pathogens, TGF-β and IFN-γ are released. Subsequently, VDR is activated to express more cathelicidin and defensin, which are known to regulate the composition of bacterial flora. Additionally, VDR is associated with TLRs, which functions to invoke the immune system to recognize and, further, to respond to bacteria[37].

Studies from our laboratory demonstrate a link between intestinal epithelial VDR and bacteria in IBD in experimental colitis models. In our work, we found profound alterations in microbiome profile not only in taxonomic classification but also in KEGG modules related to detoxification, cancer, diabetes mellitus, infections, signaling pathways,etc. (unpublished data). We have begun to understand the nature of microbiome affected by VDR expression in the intestine. On one hand, using cultured intestinal cells,Salmonellacolitis, and mono-associated commensalE.coliF18 in originally germ-free mice, we have reported that enteric bacteria activate VDR signaling[38]. On the other hand, VDR expression protects host from invasive pathogens and maintain homeostasis[38-39]. VDR-/-mice have increased bacterial loads in the intestine[35]. Deficiency in vitamin D/VDR signaling may results in defects in autophagy, compromise the integrity of mucosal barrier, and increase the vulnerability to and risks of IBD[34-40].

Interestingly, VDR, NF-κB, and TLR/NOD2 are closely associated and are all implicated in the pathogenesis of IBD[41-42]. Loss of VDR negatively regulates NF-κB activity. Vitamin D3induces NOD2/CARD15-defensin pathway[43]. Abnormalities of these key signaling pathways can perturb intestinal homeostasis and promote IBD in genetically susceptible individuals.

5 Applications that manipulate intestinal microbiome in clinical trials

Traditional therapeutics against of IBD aims to suppress the intestinal inflammation, and sometimes surgery is required, leaving the life quality of the patients considerably compromised[44]. Strategies along this line were developed to some extent during the past decade. However, they still cannot meet the urgent need for more effective therapeutic methods. On the other hand, manipulation of microbiota, due to its high efficacy under a lot of clinical scenarios, has become more and more popular for the treatment of IBD. The available approaches include fecal microbiota transplantation (FMT), pre-, pro-, syn- and post-biotics, helminthic therapy,etc., which were discussed in details in our previous review[14].

6 Current challenge and future direction

The microbial flora modulates numerous aspects of human physiology and is a critical factor in the development of IBD. Unfortunately, the research on microbiome in human diseases remains descriptive and some of the basic questions about the role of the microbiota in IBD remain unanswered: 1) Is dysbiosis a cause or consequence of diseases? 2) Are the host-microbe disturbances constant or dynamic throughout the natural history of these diseases and what role do environmental and dietary factors play in determining the risk and course of IBD? 3) Are these diseases caused by the emergence of pathobionts and/or disappearance of symbionts, or merely due to an aberrant host immune response to commensal microbiota? Insights into the microbial-host interrelationships are hampered by the limited knowledge of the diversity and complexity of microbiota. Therefore, studies on the distribution, dynamics, and functions of microbial flora in IBD will provide insights into the pathogenesis of IBD and potential therapeutic strategies.

The intestinal microbes affect barrier functions and immune system, supply key nutrients, modulate energy metabolism, stimulate cell growth, repress the growth of harmful microorganisms, and defend against diseases. The etiology of IBD has been described as interactions among environmental, genetic, microbiome, and immune factors. In the jejunum/ileum from the stomach and duodenum, and in the large intestine, colon-residing bacteria achieve the highest cell densities recorded for any ecosystem. The distribution, dynamics, and functions of intestinal microbes are closely associated with the intestinal homeostasis and contribute to the pathogenesis of IBD. The host-bacterial interactions are regulated by the signaling pathways, including TLR/NOD, NF-kB, and VDR. The flora might be a rich repository of metabolites that can be used in therapeutics against IBD.

The essence of manipulating intestinal microbiome is to reestablish the delicate balance of different bacterial populations. Therefore, it is urgent to accurately define the functional roles of specific bacterial species using mono-association models. Bacterial gene profile at functional level is similar among different individual, and hence is more meaningful than taxonomic categorization of the microbiome. Totally, more than 163 loci have been associated with IBD, including the most prominent NOD2, ATG16L1, and IL23R, and 110 of them are shared in CD and UC patients[45-46]. The interactions and co-adaption of the host and microbiome together arbitrate the fate of gut. Worth mentioning, intestinal microbiome also includes fungi, viruses and phages, which are not as frequently studied, but hold potential scientific and clinical values, and therefore would further advance therapeutic efficacy upon further exploration. Manipulation of intestinal microbiome is characterized by high cure rates and low remission, but still high quality studies with follow-up long enough are desperately needed to qualify the clinical usage.

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