MicroBiology

The Host Microbiome Regulates and Maintains Human Health: A Primer and Perspective for Non-Microbiologists

Sunil Thomas1,*, Jacques Izard2, Emily Walsh3, Kristen Batich4, Pakawat Chongsathidkiet4, Gerard Clarke5, David A. Sela6, Alexander J. Muller1, James M. Mullin1, Korin Albert7, John P. Gilligan1, Katherine DiGuilio1, Rima Dilbarova1, Walker Alexander1, and George C. Prendergast1

1Lankenau Institute for Medical Research, Wynnewood, PA, USA 2University of Nebraska– Lincoln, Department of Food Science and Technology, 1901 North 21 Street, Lincoln, NE, USA 3Harvard School of Dental Medicine, Department of Oral Medicine, Infection and Immunity, 188 Longwood Avenue, Boston, MA, USA 4Duke Brain Tumor Immunotherapy Program, Department of Neurosurgery, Department of Surgery; Department of Pathology, Duke University Medical Center, Durham, NC, USA 5Department of Psychiatry and Neurobehavioural Science, APC Microbiome Institute University College Cork, Cork, Ireland 6Department of Food Science, University of Massachusetts, Amherst, MA; Department of Microbiology, University of Massachusetts, Amherst, MA; Center for Microbiome Research, University of Massachusetts Medical School, Worcester, MA, USA 7Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, MA; Department of Food Science, University of Massachusetts, Amherst, MA, USA

Abstract

Humans consider themselves discrete autonomous organisms, but recent research is rapidly

strengthening the appreciation that associated microorganisms make essential contributions to

human health and well-being. Each person is inhabited and also surrounded by his/her own

signature microbial cloud. A low diversity of microorganisms is associated with a plethora of

diseases including allergy, diabetes, obesity, arthritis, inflammatory bowel diseases and even

neuropsychiatric disorders. Thus, an interaction of microorganisms with the host immune system

is required for a healthy body. Exposure to microorganisms from the moment we are born and

appropriate microbiome assembly during childhood are essential for establishing an active

immune system necessary to prevent disease later in life. Exposure to microorganisms educates the

immune system, induces adaptive immunity and initiates memory B and T cells that are essential

to combat various pathogens. The correct microbial-based education of immune cells may be

critical in preventing the development of autoimmune diseases and cancer. This review provides a

Author for correspondence: Sunil Thomas, Lankenau Institute for Medical Research, 100 E. Lancaster Ave., Wynnewood, PA 19096, Phone: 484-476-3507, Fax: 484-476-2205, [email protected] and [email protected].

Conflicts of interest: Conflicts of Interest: E. Walsh is an employee of Seres Therapeutics. D.A. Sela has ownership interest in a patent (2010/0113383 A1) and receives honoraria for speaking engagements. G.C. Prendergast has a commercial research grant from Janssen Pharmaceuticals, ownership interest in New Link Genetics Inc., Meditope Biosciences Inc., Dynamis Pharmaceuticals, and Man's Best Friend Therapeutics, and serves on consultant/advisory boards for New Link Genetics Inc., Ribonova Inc., Kyn Therapeutics Inc., Vitae Pharmaceuticals, Biogen Inc., OrbiMed Advisors LLC, and Guidepoint Global LLC. No potential conflicts of interest were disclosed by the other authors.

HHS Public Access Author manuscript Cancer Res. Author manuscript; available in PMC 2017 October 15.

Published in final edited form as: Cancer Res. 2017 April 15; 77(8): 1783–1812. doi:10.1158/0008-5472.CAN-16-2929.

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broad overview of the importance of the host microbiome and accumulating knowledge of how it

regulates and maintains a healthy human system.

Keywords

Microbiome; host; human health; disease; infant; cancer

Role of the microbiome in maintaining host health

At all stages of life, humans are associated with microorganisms and their products. Humans

co-evolved with microbes in the environment, and each body habitat has a unique set of

microorganisms in its microbiota (1). The microbiome (term coined by Joshua Lederberg)

consists of the ecological community of commensal, symbiotic, and pathogenic

microorganisms that share our body (2). The host organism together with its microbiome

constitutes the “holobiont” (Greek, holos, whole/entire), and the totality of the genome is the “hologenome” (3,4). Changes in the holobiont may impact the complex signaling network

thereby influencing the hologenome leading to health or disease.

The human body is estimated to be composed of 3 × 1013 eukaryotic cells and 3.9 × 1013

colonizing microorganisms, such that host cells and microbiota are of nearly the same

number in an individual (5). The largest concentrations of microbes occupy the gut, skin and

oral cavity. The microbiota of our system is well tolerated by our immune system due to the

co-evolution of these microorganisms over time. The overwhelming majority of gut

microbiota are Eubacteria. The collective genome, or metagenome, of the enteric microbiota

contains over 100 times the number of genes in the human genome, and there are

approximately 10-fold more genes in each of our microbiomes than in each of us, encoding

the greatest source of potential antigens with which the immune system must cope (6). The

microbiome in humans significantly enriches the metabolism of glycans, amino acids and

xenobiotics. It is also responsible for the synthesis of vitamins, isoprenoids and other

nutrients making humans “super organisms” whose metabolism represents an amalgamation

of microbial and human attributes (7).

Each individual emits a distinct and personalized cloud of microorganisms into his or her

surroundings (8). The microbiome in humans is also not constant during lifespan, but rather

changes with age. Culture and location also have a profound impact on the microbiome (9).

Health status is yet another factor influencing the microbiome compositional status. In one

study, the growth dynamics of gut microbiota in health and disease have been inferred from

a single metagenomic sample (10). These authors copy the number and ratio at origin and

terminus to detect the actively growing species in a microbiome. In this way, they showed

differences between virulent and avirulent strains, population diurnal oscillations and

bacterial species predominant during disease and diet changes.

Diet is a particularly important factor in determining the microbiota composition of the gut

(11). Thus vegans, vegetarians and omnivores have distinct microbiomes. Total counts of

Bacteroides spp., Bifidobacterium spp., Escherichia coli and Enterobacteriaceae spp. were significantly lower in vegan samples than in controls. In contrast, total counts of Klebsiella

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spp., Enterobacter spp., other Enterobacteriaceae, Enterococcus spp., Lactobacillus spp., Citrobacter spp. and Clostridium spp. were similar in people with different diets. Subjects on a vegetarian diet ranked between vegans and omnivores. The total microbial count did not

differ between the dietary groups (12). Notably, the microbiome of a person can be altered

rapidly by changes in dietary patterns. It has been demonstrated by David et al. (13) that

short-term consumption of diets composed entirely of animal or plant products can alter the

microbial community structure. An animal-based diet increased the abundance of bile-

tolerant microorganisms, including Alistipes, Bilophila and Bacteroides and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia spp, Eubacterium rectale and Ruminococcus bromii). Thus, the gut microbiome rapidly responds to diet.

Microbiome taxonomy and its future

The taxonomy of microbiomes reflects their complexity and the challenges encountered in

their understanding. Microbiomes include species across all major kingdoms, including

viruses as well as Archaea, bacteria and microbial eukaryotes. Our present depth of

knowledge is associated with different methods of investigation, targeted surveys and scope

of studies conducted. To date, the most comprehensively investigated phylogenic group in

health and disease has been bacteria.

Prokaryotes

List of Prokaryotic Names with Standing in Nomenclature (LPSN; http://www.bacterio.net)

includes two prokaryotic domains (or empires), subdivided into 30 phyla in the domain

Bacteria and 5 phyla in the domain Archaea. Together these 35 phyla encompass about

2,400 genera and 12,400 species (14). This list is based on strict requirements, including the

availability of reference strains, and does not include all available reference strains deposited

in culture collections, including the ones for which genome sequences are already available

(15-17). The addition of whole-genome phylogenetic analysis allows a refined positioning in

the phylogenetic hierarchy as new tools are being developed (18-21). This approach brings

some conflicts with the present classification, as has happened when the 16S rRNA gene

phylogenetic classification competed with the phenotypic classification (22-24).

Additionally the ability to target and obtain the sequence of the genes used for phylogenetic

classification (16S rRNA, recA, rpoB, gyrB etc.) using culture-independent methods also

adds to the known diversity. Specialized curated databases that allow the propagation of this

knowledge include SILVA, Ribosomal Database Project and the Human Oral Microbiome

Database (23,25,26). This culture-independent approach raised to 46 the number of phyla

(23). How much of this diversity is in the human microbiome is unclear. However, it is

already clear that organisms known to be environmental are also associated with health, and

that at least 30 prokaryotic phyla and 950 genera are associated with the human microbiome

(27,28).

Microbial eukaryotes

The microbial eukaryotes are extremely diverse and do not fit under a single keyword. While

accurate, the eukaryotic supergroups defined by phylogenomics (Opisthokonta, Amoebozoa,

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Excavata, Archaeplastida and SAR [Stramenopiles+Alveolates+Rhizaria]), are unfortunately

uninformative compared to previous classification methods used in the literature. From the

clinician to the lay person, terms such as fungi, protists, parasites, protozoa and amoebae are

much more familiar. In this area, present knowledge is based mostly on their roles as

causative agents of disease; few studies have focused on healthy individuals or within a

defined illness in a restricted number of individuals (29-33). Until recently, the focus on

single disease agents also meant neglecting the reminder of the eukaryotic microbiome

(34,35).

The human eukaryotic microbiome includes pathogens, commensals and beneficial

organisms. The fungi (Opisthokonta) harbor a wide diversity of organisms, with an overlap

for the skin with the local environment (35). The fungi include the Ascomycota (Candida albicans), Basidiomycota (Cryptococcus neoformans), Microsporida (Encephalitozoon intestinalis), Zygomycota (Rhizopus microsporus). As fungi are part of the environment and human alimentation, it may be difficult to differentiate between transient and commensal

organisms without a longitudinal study, unless a disease or an opportunistic infection occurs

(36,37). The Acanthocephala, most closely related to the rotifers, include

Macracanthorhynchus hirudinaceus. The helminths (Opisthokonta), which are classified as part of the animals, include the cestodes (tapeworms: Taenia saginata), trematodes (flukes: Schistosoma mansoni), and nematodes (roundworms: Enterobius vermicularis). While the majority of the helminths cause illness in millions of people worldwide, a few helminth

species have been used in therapy (38,39). The protozoa include the Amoebozoa

[Amoebozoa] (amoeba: Entamoeba histolytica), Metamonada [Excavata] (flagellates: Giardia intestinalis), Parabasilia [Excavata] (Dientamoeba fragilis), Ciliophora [SAR] (ciliates: Ballentidium coli), Apicomplexa [SAR] (Cryptosporidium parvum) and Stramenopile [SAR] (Blastocystis hominis). These protozoa are all medically important even though not all carriers are symptomatic (30,35). The Archaeplastida (including green and

red algae) can be present in the microbiome of the skin and digestive tract. Additional

sequences available through the sequencing of targeted genes including via metagenomics

has expanded this knowledge and is maintained and curated in databases such as SILVA

(23). A current view of the tree of life, encompassing the total diversity represented by

sequenced genomes was published recently by Hug et al. (40).

Viruses

The gut microbiome includes bacteriophages that influence the bacterial hosts. The

bacteriophage in the human gut are of three classes: a set of core bacteriophages shared

among more than half of the human population, a common set of bacteriophages found in

20%–50% of individuals, and a set of bacteriophages that are unique to a person. Healthy

gut phageome (aggregate of bacteriophage in the gut) is significantly decreased in

individuals with gastrointestinal diseases (41). The International Committee on Taxonomy of

Viruses in its 2014 release listed 104 families, 505 genera and 3,186 species of all known

viruses (42). The human virome overlaps with other animal viromes. These dsDNA, ssDNA,

dsRNA, ssRNA- ssRNA+ viruses and dsDNA and ssRNA retroviruses can affect any of the

tissues within the body. Human protists (non-fungal microbial eukaryotes) have their own

viral challenges which are being uncovered within the human virome (43). Much more is

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known about mycoviruses and their intracellular transmission during cell division and

sporogenesis and it is recognized that their life cycles generally lack an extracellular phase.

Most known mycoviruses have dsRNA genome, but an increasing number of positive- or

negative-strand ssRNA and ssDNA viruses have been isolated and characterized (44). Most

of the archaeal viruses have been isolated from members of the Euryarchaeota and

Crenarchaeota with broader morphological characteristics than their bacterial counterpart

(45). Little is known of the impact of archaeal viruses on the human virome. The dsDNA,

ssDNA, dsRNA, ssRNA bacteriophages have a great impact on prokaryotic ecology through

their ability to modify population structure. A list of viral pathogens is maintained by the

ViPR resource, while prophages are available at PHAST (46,47).

Future of taxonomy

An open challenge in taxonomy is to refine the classifications to be more compatible with

the emerging methods of molecular bio-surveillance and detection requiring targets

associated to an outcome or being able to identify strains at multiple body-sites across the

domains of life (21,48). This work is dependent on a greater understanding of the true

diversity in the population with the direct sequencing of large sample sets and/or large

cohorts such as the Human Microbiome Project, MetaHIT, BioMarks, and future large scale

projects (49-51). New resources are now attempting to bridge the different approaches and

topics of detection across domains, such as the Human Pan-Microbe Community database

(HPMCD) (34). The ability to isolate and sequence single cells offers the opportunity to

deepen both our understanding of the genomic composition of taxonomic diversity, as well

to put this diversity in context of its environment, microbial partners, biogeography, and host

physiologic status both at the local and systemic levels (52-54).

Beyond the present time taxonomy and the species level classification focused on vertical

transmission of conserved information to descendant cells. The strain definition is associated

with gene composition and gene modification including mutations and antigenic variation

following homologous DNA recombination, CRISPR system (Clustered Regularly

Interspaced Short Palindromic Repeats), gene transfer, mobile elements, epigenetics, etc.

Moving from targeted-gene phylogeny, to whole-genome comparison has its own limitations

that can be complemented by the inclusion of other omics as once metabolic panels, protein

and DNA finger-printing profiles were used. This polyphasic analysis allows understanding

genetic relatedness and phylogenic relationship in the context of disease, reservoir, niche

transmission within a single individual, propagation within a population and dissemination

among environments. An increasing number of gene transfer events among domains are

being documented as well as across ecological niches (55-57). The genetic modification can

be due to increases in genetic content but genetic loss also has critical consequences in

competitiveness or niche settlement (58,59). As no clear general phylogenetic definition of

strain has emerged in this era of genomics, efforts are to differentiate the different isolates

with markers not yet found in other genomes and/or single nucleotide polymorphism

(20,21,60,61).

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The infant microbiome and transgeneration effects

Until recently the placenta was considered a “sterile” intrauterine environment. Aagaard et

al. (62) reported that the placental microbiome is consistently different from other parts of

the body, including the skin and urogenital tract. Interestingly, the placental microbiome is

most similar to that of the oral cavity. Thus, these authors suggested that microbes travel to

the placenta from the mouth via the blood. The results reinforce data that have suggested a

link between periodontal disease in the mother and the risk of preterm birth.

Infant-associated microbial communities initially possess high concentrations of facultative

anaerobes such as Escherichia coli and Streptococcus spp., but these populations are replaced by strict anaerobes coinciding with a reduction in oxygen tension (63-65). In

addition to environmental routes of inoculation, the specific mode by which the infant is

delivered is now known to influence the early gut microbiome structure and trajectory.

Although there is emerging evidence that the fetus encounters placental and amniotic

bacteria in utero, it is clear that parturition contributes to the infant's first major inoculation of colonizing microbes (63,66,67). Microbial communities colonize external surfaces of the

infant immediately following birth. This includes various microbial populations that are

established and maintained along the gastrointestinal tract (GIT). The percentage of babies

delivered through cesarean section has risen in many countries. While a number of cesarean

section deliveries are performed for obstetrical indications, a large proportion is not

medically indicated and may be due to maternal request and may incur several risks for the

child (68). Obstetricians in many medical settings are paid more for cesarean delivery and it

is well known that private hospitals and practitioners encourage cesarean delivery (69).

However, recent studies demonstrated that babies born vaginally are healthier compared to

babies born by cesarean delivery. As such, infants delivered vaginally tend to harbor

microbiota that are typically encountered in the female reproductive tract, such as

Lactobacillus. In contrast, cesarean delivery is typically associated with Staphylococcus spp. and other bacteria that are associated with the mother's skin and hospital environment

(65,70,71). Children delivered by cesarean section have significantly increased risk of

asthma, systemic connective tissue disorders, juvenile arthritis, inflammatory bowel disease,

immune deficiencies, leukemia (72) and Crohn's disease (73). Although there are some

indications that infants born via cesarean section may be more susceptible to colonization by

Clostridium difficile or methicillin-resistant Staphylococcus aureus (MRSA), and may be at an increased risk for pathologies later in life (71,72), additional mechanistic studies are

required to conclude causal relationships in this regard.

The infant microbiome exhibits several shared attributes regardless of birth method. In

general, the infant microbiome is often dominated by the genera Bifidobacterium, Bacteroidetes, and members of clostridial taxa (74). In the seminal 2012 study conducted by Yatsunenko et al. (9) gut microbiomes were characterized from individuals located in three distinct geographic locations (i.e., United States, rural Malawi, and rural Venezuela).

Regardless of the host's location microbial populations converge toward an adult community

by three years of age (9). Furthermore, microbial community diversity increased as the host

aged across all populations (9). Infant microbiomes exhibit hallmarks of functional

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redundancy, in that inter-individual taxonomic variation is common despite sharing a stable

and uniform metabolic potential (75). This functional redundancy during neonatal

development may contribute to metabolic, digestive and immune system homeostasis

(74,76). During infancy, the impact of alterations in community assembly on function has

been linked to outcomes such as malnourishment, C. difficile-associated diarrhea, and necrotizing enterocolitis (74,76,77). Early life microbiome disruption may potentially

increase risk for developing celiac disease, asthma, type-1 diabetes, and obesity

(70,72,78-81). These conditions could have long-term medical implications that interact

reciprocally with the gut microbiome.

Perturbations or durable disruptions of the infant microbiome may proceed via several paths,

with hospitalization and antibiotic use considered to be primary causes. Pre-term and term

infants who are hospitalized early in life are at a greater risk for nosocomial C. difficile infection (74,82). Thus the hospital environment is a reservoir for infectious agents that may

be deleterious for at-risk populations such as pre-term infants with underdeveloped

immunological function. As antibiotics select for resistant and resilient strains,

indiscriminant usage of antimicrobials may drive gut dysbiosis in certain instances. Several

studies have characterized the influence of antibiotic usage on restricting gut microbiota

diversity, potentially increasing susceptibility to aggressive bacterial infections like C. difficile and vancomycin-resistant Enterococcus bacteremia (82-84).

As it does in adults, diet exerts a strong influence on the structural composition of infant-

hosted microbiomes. Culture-dependent and independent studies indicate that breastfed

infants often possess a significantly different and less diverse gut microbiome relative to

formula-fed infants (9,64,85-87). Accordingly, human milk incorporates several bioactive

compounds important for infant nutrition, including lipids, proteins and lactose. In addition

several milk molecules enhance immunological and neurological development (88-90).

Escaping digestion by host glycosyl hydrolases, soluble milk glycans are transferred to the

distal colon where they are exposed to the gut microbiota of the infant. Thus these human

milk oligosaccharides (HMO) are available to guide the establishment and function of the

infant microbiome. HMOs are heterogeneous carbohydrate polymers that are the third most

abundant milk component at several grams per liter (91). HMO structures incorporate the

monosaccharides D-glucose, D-galactose, N-acetylglucosamine, L-fucose and N-

acetylneuraminic acid, with over 200 unique HMO isomers composed of these components

identified to date (91,92).

Breastfeeding infants often display a microbiome enriched for commensal Bifidobacteria that can utilize HMOs. Bifidobacteria are Gram-positive anaerobes of the phylum Actinobacteria which typically colonize infants and adults to lesser degree (93-95). Accordingly, Bifidobacterium longum subsp. infantis is a major commensal of breastfed infants with this lineage possessing a large genomic cluster that enables HMOs utilization

(95). That unique gene assemblage permits the catabolism of specific small mass HMOs that

other Bifidobacteria cannot process. For example, in comparison to other Bifidobacteria, B. longum subsp. infantis flourishes in the presence of milk that contains α1,2-fucosylated HMOs (96). An individual's complement of HMO structures is somewhat dependent on the

mother's genotype and may vary by gestational age and stage of lactation. The relative

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concentrations of α1,2-linked fucosyl moieties depend on the fucosyltransferase 2 (FUT2) allele (96). Women with a functional copy of this gene, termed secretors, may confer certain health benefits to their infant such as a decreased risk for diarrheal diseases (96-98). HMOs

can decrease the presence of gastrointestinal pathogens using two primary mechanisms.

HMOs themselves mimic pathogen binding sites of receptors that decorate the surface of

host cells (99-101). Studies have documented this effect using Vibrio cholerae, Streptococcus pnemoniae and Escherichia coli (102-104). In addition, high levels of Bifidobacteria are correlated with lower incidence of potentially dangerous neonatal infections, potentially due to competitive exclusion (105,106). Gut microbiota development

during infancy can have long-lasting effects on the individual's future health. Colonization of

fucosyllactose (FL)-utilizing bifidobacteria is due to an ABC transporter that acts as a key

genetic factor for FL utilization (107).

Human milk is generally accepted as the best nutrition for newborns and has been shown to

support the optimal growth and development of infants (108). Human milk also provides

bioactive components that are important to optimize gut microbial colonization, immune

maturation, metabolic development and even cognitive development. Breast milk has a low

buffering capacity which would make the gut more susceptible to a lowering of pH due to

acid production from bacterial fermentation in the colon. The fecal pH of the breast-fed

infant is between 5 and 6 dominated by Bifidobacteria, whereas formula-fed infants have a fecal pH in the range of 8–9. The acetic acid in the gut of breast-fed infants is frequently

present as an acetate buffer. This effect was not observed in formula-fed infants. The lower

pH in the gut is an important factor in restricting the growth of Enterobacteria, Clostridia

and the Bacteroides, and favors the proliferation of the acid-tolerant Bifidobacteria and Lactobacilli (109). Human milk also contains many antimicrobial factors, such as partially digested or fermented peptides, milk-borne fatty acids, human lactoferrin, lysozyme, and

secretory IgA (sIgA). These factors may decrease the prevalence of pathogens in the gut's

ecosystem in infants. The broad range of nondigestible oligosaccharides specifically found

in human milk but not in other mammals' milk (108) is a major factor in the prevention of

pathogen growth in the gastrointestinal tract. Stunted infants fed poorly have low amounts of

sialylated HMOs in the gut. These oligosaccharides are not used by the body, but rather used

by the gut microbes. Charbonneau et al. (110) colonized germ-free mice with a consortium

of bacterial strains cultured from the fecal microbiota of a 6-month-old stunted infant and

fed recipient animals with normal diet with or without purified sialylated bovine milk

oligosaccharides (S-BMO). S-BMO produced a microbiota-dependent body weight gain

indicating growth promotion in the presence of gut microbiota. However control animals

that were germ free did not increase body weight, demonstrating some bacteria in the gut are

involved in weight gain.

Infant formula is often based on bovine milk unless it is plant derived. Fluid dairy milk

contains oligosaccharides with similar structure to HMOs, which may suggest similar

functionality despite being incorporated at relatively low concentrations (108,111-113). At

the moment, there are efforts to supplement infant formula with oligosaccharides although

HMO structures are difficult to synthesize and may not be commercially viable (114).

However, oligosaccharides from other sources may increase bifidobacterial concentration as

a preferred endpoint, including galacto-oligosaccharides and fructo-oligosaccharides.

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Use of oral probiotics by the mother during pregnancy is thought to help the developing

baby. Microbes in placenta or amniotic fluid affect fetal innate immune gene expression

during late pregnancy. Maternal probiotic supplementation significantly modulated the

expression of TLR-related genes both in the placenta and in the fetal gut. Thus, fetal and

placental immune physiology may be modulated by maternal dietary interventions including

using specific probiotics (115,116). It has also been shown that maternal probiotic

supplementation during pregnancy and breastfeeding reduces the risk of eczema in the infant

(116). Probiotic supplements continue to impact infants in their early years. It has been

shown that infant formula supplemented with the probiotics Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb-12 offer a safe means of reducing the risk of early acute otitis media and antibiotic use and the risk of recurrent respiratory infections during the first year

of life (117). Probiotics enhance gut-specific IgA responses, which are frequently defective

in children with food allergy (118). Kainonen et al. (119) have demonstrated that exclusive

breast-feeding promotes an anti-inflammatory cytokine milieu, which is maintained

throughout infancy. Such an immunological environment limits hyper-responsiveness and

promotes tolerization, thereby prohibiting the onset of allergic disease.

Infantile colic (excessive crying), is a common problem in about 20% of healthy thriving

infants in the first three months of life (120). The risk factors associated with the

development of infantile colic include, maternal smoking, increased maternal age and

firstborn status. Infantile colic could also be related to cow's milk protein allergy and atopy

(121). Several studies have demonstrated that administration of probiotics containing

Lactobacillus reuteri DSM 17938 significantly improved colic symptoms by reducing crying and fussing times in breastfed infants with colic (122,123). Treatment of colic with L. reuteri did not affect the global composition of the microbiota. The decrease in colicky symptoms

was linked to changes in the microbiota, with a relative increased abundance of the phyla

Bacteroidetes and genus Bacteroides after treatment with L. reuteri (124).

Microbiome and aging

As humans age, the composition of the microbiome also changes (9). Aging is accompanied

by the onset of a myriad of clinical changes, including a basal proinflammatory state

(“inflamm-aging”) that directly interfaces with the microbiota of older adults and enhances

their susceptibility to diseases that accompany aging. Studies in older adults demonstrate

that the gut microbiota correlates with diet, basal level of inflammation and location of

residence (e.g., community dwelling, long-term care settings) (125-127). Links between the

microbiota and a variety of clinical problems plaguing older adults have been made,

including physical frailty, Clostridium difficile, colitis, vulvovaginal atrophy, colorectal carcinoma and atherosclerotic disease (128).

The most drastic change associated with the aging gut is a change in the relative proportion

of organisms, e.g., Firmicutes dominate in the young and Bacteroidetes in the elderly. Reduction in the diversity of bacteria comprising subpopulations is seen in individuals with

high frailty, although living in a community undermines this alteration (125,129).

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Aging-associated oxidative stress induces aggressive potential and virulence factors of

anaerobic bacteria, thereby causing morphologic alterations of bacterial cells that could

impact the host. The microbiota may also influence host gene expression by regulating

microRNAs (130). Analysis of the network functions revealed that differentially regulated

miRNAs between infants and adults and miRNAs that decreased during aging shared two

network functions: inflammatory disease and inflammatory response. miRNAs promote

aging by modulating their targets to drive cell senescence and aging in different tissues or

organs. There is significant variation in the expression of miRNA during aging. Genome-

wide assessment of miRNA expression revealed that the majority of miRNAs decreased with

age (131,132). Interestingly, host-derived miRNAs may also influence the composition of

the gut microbiome (133).

It has been documented that calorie restriction can increase the life span of model organisms

(134). Notably, Zhang et al. (135) demonstrated that calorie restriction enriches bacterial

phylotypes positively correlated with lifespan. Bacteria of the genus Lactobacillus have been shown to increase in animals on low-fat diet, and this environment reduces phylotypes which

are negatively correlated with lifespan. Caloric restriction-induced changes in gut microbiota

occur concomitantly with a significant reduction in serum levels of lipopolysaccharide-

binding protein (LPS), suggesting that animals undergoing calorie restriction establish a

structurally balanced architecture of gut microbiota that exerts a health benefit through the

reduction of antigen load from the gut. Strikingly, dietary changes can detectibly influence

host environment in as little as 24 hours, with longer term changes correlating with novel

enterotype clustering in the host (136).

Multiple studies in centenarians indicate extreme aging is characterized by microbial

changes deemed unique from other age groups, with emphasis placed on organismal

composition and increased inflammatory effects (137,138). Fecal sampling by Rampelli et

al. (138) revealed a distinctive functional profile with a decrease in short chain fatty acid

production and saccharolytic potential but an increase in proteolytic potential. One hundred

and sixteen microbial genes were found to be correlated with aging, including those

essential to the metabolism of tryptophan, phenylalanine, tyrosine, valine, and lysine.

Implications of such variability include changes in wellbeing, aging, and disease

susceptibility. This was accompanied by an increase in occurrence of pathobionts, bacteria

usually present in low numbers in the adult. Pro-inflammatory effects of the pathobionts are

exaggerated by a decrease in Faecalibacterium prausnitzii, a species associated with anti- inflammatory influences (137).

The relationship between aging and the microbiome is not strictly one-sided; it has been

demonstrated that host aging can actually be impacted by interspecies communication.

Animal fecundity, development time and lifespan were all dependent on the amount and type

of bacteria they were fed. There are multiple lines of evidence demonstrating the ability of

microbes to substantially change host physiology as it pertains to these parameters (139).

Accordingly, manipulating the microbiome of older adults holds promise as an innovative

strategy to positively influence the development of co-morbidities associated with aging

(128).

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Rozsa et al. (140) recently proposed the ‘microbiome mutiny hypothesis’, whereby some

microorganisms of the microbiome could switch to higher virulence (microbiome mutiny) in

old or seriously ill people, to optimize their transmission under the conditions of increased

background mortality. This proposed virulence shift might contribute to the death of old or

seriously ill people even in the absence of apparent disease.

In the central nervous system (CNS), polyphenols present in many edible plants exert anti-

inflammatory effects (141) and act on the brain in several ways. Like antioxidant vitamins,

dietary polyphenols contribute to the regulation of oxidative stress and improve vascular

health. Notably, intestinal microbiota convert dietary polyphenols to phenolic acids,

stimulating proliferation of Bifidobacteria and decreasing the ratio of Firmicutes to Bacteroidetes, relative to controls. Polyphenols also stimulate short chain fatty acid production by bacteria (142). Wang et al. (143) recently reported that the microbiome can

convert grape-derived polyphenol to the phenolic acids 3-hydroxybenzoic acid and 3-(3′- hydroxyphenyl)propionic acid, which interfere with the assembly of β-amyloid peptides into neurotoxic β-amyloid aggregates that play key roles in the pathogenesis of Alzheimer's disease (AD). Thus, in the brain and other tissues, many healthful effects of polyphenols

may relate to their conversion to various metabolic derivatives by the gut microbiome while

aging.

Microorganisms and immune function

Through co-evolution with their hosts, microbes exert a major influence in shaping the

development of the immune system, putting it under selective pressure to develop the

capability to discern between invasive pathogens that it is imperative to control and

commensal resident microbes that are beneficial to tolerate (144,145). Many developmental

aspects of the adaptive immune system are influenced by the composition of bacterial

colonization of the gut. Thus, the mammalian immune system, which is tasked with the duty

of controlling microorganisms, is in turn fundamentally shaped by microorganisms (146).

For example, it has been demonstrated that changes to the symbiotic microbiota early in life,

or the absence of it, can lead to exacerbated type 2 immunity and allergies due to aberrant

immune functionality. The microbiota is a strong inducer of proinflammatory T helper 17

(TH17) cells and regulatory T cells (Treg) in the intestine. The microbiota-induced Treg express the nuclear hormone receptor RORγt and differentiate along a pathway that also leads to TH17 cells. In the absence of RORγt+ Treg, TH2-driven defense against helminths is more efficient, whereas TH2-associated pathology is exacerbated. Thus, the microbiota

regulate type 2 responses through induction of type 3 RORγt+ Treg and TH17 cells, thereby balancing immune responses at mucosal surfaces (147). Exercise can also influence the

immune system and how they modulate microorganisms (148,149). Intense exercise causes

immunosuppression, while moderate intensity exercise improves immune function and

potentially reduces risk and severity of respiratory viral infection by increasing stress

hormones, reduce excessive local inflammation and skew the immune response to a Th2

phenotype (148). Similarly, exercise can also influence bacterial infections. Pape et al. (150)

demonstrated a reduction of bacterial infection in people with physical activity compared to

those that maintain a sedentary lifestyle.

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Similar to adaptive immunity, the innate immunity is also influenced by the microbiome.

One example of this is neutrophil aging. Aged neutrophils exhibit impaired migration and

reduced pro-inflammatory properties. Microbiota influence neutrophil aging via Toll-like

receptor and MyD88-mediated signaling pathways. Depletion of the microbiota significantly

reduces circulating numbers of aged neutrophils and improves pathogenesis and

inflammation-related organ damage in models of sickle-cell disease or endotoxin-induced

septic shock. Thus, host microbiota may play a role in regulating a disease-promoting

neutrophil subset that promotes tissue injury in various inflammatory diseases (151).

While active immunity is essential to combat infection, inadequate control over immune

responsiveness due to the inability to establish immune tolerance can also have dire

consequences, regardless of whether the response is directed against a foreign pathogen or

self. Meanwhile, one of the major benefits of immune tolerance is the ability to maintain a

commensal microbiome consisting of a multitude of foreign microorganisms. Thus, the

mechanisms for establishing tolerance are a vital aspect of the immune regulatory

framework. One crucial element in instructing the immune system to be self-tolerant is the

education of thymus T cells during development. In the thymus, self-reactive cells are either

eliminated or differentiated into tolerogenic Foxp3(+) regulatory T cells (Treg) (152). Apart

from the thymus, the immune system is also educated in the gut where it has been shown

that the interaction of T cells with commensal microbiota results in the peripheral generation

of Treg rather than pathogenic effector cells. Failure of this tolerogenic process can lead to

the development of autoimmune diseases including colitis (152).

Microorganisms encountered early in life prevent autoimmunity and allergy

The human microbiome is important for human health, behavior and disease, yet its function

and dynamics during healthy and disease states are not fully understood (153). It is also not

fully understood how the microbiome interacts with the host immunity thereby preventing

autoimmunity. The hygiene hypothesis first put forward by Strachan (154) postulates that the

lack of early exposure to microorganisms (either beneficial or pathogenic) would lead to the

poor development of the immune system. The leading idea is that some microorganisms that

co-evolved with us are able to protect against a large spectrum of immune-related disorders

(155). While the hygiene hypothesis is not universally applicable, it offers some explanative

power to interpret the effects of microorganism exposure in early life on preventing

autoimmunity and allergy.

Children growing up on dairy farms are protected from allergy, hay fever, and asthma

(156,157). Asthma is a chronic inflammatory disease triggered by acute inflammation of the

bronchial tube leading to production of extra mucus. This can make breathing difficult and

trigger coughing, wheezing and shortness of breath. The number of asthma cases is

increasing all over the world, but the causes remain obscure. It has been hypothesized that

increased cleanliness, reduced family size and subsequent decreased microbial exposure

could explain the increases in global asthma prevalence (158). Evidence from bronchial

brushings implicates phyla present in healthy individuals with variation present in disease

conditions such as cystic fibrosis, chronic obstructive airways disease and asthma. The

microbiome can exacerbate the phenotypes seen in the condition, as well as explain the

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variability in phenotypes observed (159). Many cytokines and chemokines are involved in

the pathophysiology of asthma. Th2 cytokines may play an important role in the

pathophysiology of asthma. The Th1 cells secrete IL-2 and interferon-γ, whereas the cytokines, IL-4, IL-5, IL-9 and IL-13, are derived from T helper type 2 (Th2) cells, although

they may also be derived from other cell types. The distinction between Th1 and Th2 cells is

not as distinct in humans as in mice (160,161). According to the hygiene hypothesis, the

lack of infection and exposure to environmental endotoxins may alter the balance between

Th1 and Th2 cells.

Though children on farms are much less likely to get asthma the underpinnings of protection

is not clearly understood. Early-life contact with livestock and their fodder, and consumption

of unprocessed cow's milk have been identified as the most effective protective exposures.

Studies of the immunobiology of farm living point to activation and modulation of innate

and adaptive immune responses by intense microbial exposures (162). Schuijs et al. (157)

demonstrated that chronic exposure to low-dose endotoxin or farm dust protects mice from

developing house dust mite (HDM)-induced asthma. Endotoxin reduced epithelial cell

cytokines that activate dendritic cells (DCs), thus suppressing type 2 immunity to HDM.

Loss of the ubiquitin-modifying enzyme A20 in lung epithelium abolished the protective

effect. A single-nucleotide polymorphism in the gene encoding A20 has been associated

with allergy and asthma risk in children growing up on farms. Thus, the farming

environment protects from allergy by modifying the communication between barrier

epithelial cells and dendritic cells through A20 induction.

From delivery, the microbiome assembly might influence asthma. Babies born via cesarean

section, who experience an altered trajectory of microbiome assembly, are more prone to

asthma than those born vaginally. Similarly, children treated with antibiotics are also more

prone to asthma attack (163). Lif Holgerson et al. (164) analyzed the oral biofilm in healthy

three-month-old infants born by cesarian section or delivered vaginally. Among over 300

bacterial taxa analyzed, Slackia exigua was detected only in infants delivered by C-section. Further, significantly more bacterial taxa were detected in the infants delivered vaginally (79

species/species clusters) compared with infants delivered by cesarean section (54 species/

species clusters). Overall, the vaginally delivered infants had a higher number of bacterial

taxa. A higher prevalence of salivary Streptococcus salivarius, Lactobacillus curvata, Lactobacillus salivarius, and Lactobacuillus casei was detected in infants delivered vaginally (165).

A longitudinal human study by Arrieta et al. (166) reported that infants at risk of asthma

have transient gut microbial dysbiosis during the first 100 days of life. The authors collected

stool and urine samples from more than 300 babies at 3 months and 1 year-old, as well as

information on their health at 1, 3, and 5 years. Then, they analyzed levels of gut microbes in

each stool sample. Babies that had low or undetectable levels of four bacteria—Lachnospira, Veillonella, Faecalibacterium, and Rothia—at 3 months old all went on to show early signs of asthma—wheezing and skin allergies—at 1 year old. The babies that did not develop

these symptoms invariably had high levels of the four microbes in their 3-month stool

samples. The authors also used the stool samples from the asthma-prone 3-month-olds to

colonize the guts of mice that had been raised in a bacteria-free environment. The animals

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developed inflamed lungs indicative of asthma. However, upon inoculating the four missing

microbes to the digestive tracts of these mice along with the feces, they no longer had a

heightened risk of developing asthma. The studies demonstrated that certain bacterial

species that are encountered early in life could train the immune system to prevent asthma.

Microbial dysbiosis in early life can alter the trajectory of immune development and provide

the setting for allergic disorders in later life (167). Dysbiosis may trigger autoimmune

diseases via inappropriate post-translational modification of host proteins (168). Endogenous

and microbial enzymes have the capacity of intestinal enzymatic neo-antigen generation by

post-translational modification of host proteins. The hygiene hypothesis stipulates that

microbial exposure during early life induces immunologic tolerance via immune stimulation,

and hence reduces the risk of allergy development. Several common lifestyle factors and

household practices, such as dishwashing methods, may increase microbial exposure.

Hesselmar et al. (169) investigated whether lifestyle factors are associated with allergy

prevalence. The authors demonstrated that in families that used hand dishwashing, allergic

diseases in children are less common than in children from families who use machine

dishwashing. The authors were of the opinion that a less-efficient dishwashing method may

induce immune tolerance via increased microbial exposure.

Autoimmunity is more prevalent in the population of some northern European countries

such as Finland and Estonia when compared to Russia. It was found that Bacteroides species are less abundant in Russians but dominate in Finnish and Estonian infants. Their

lipopolysaccharide (LPS) exposures arose primarily from Bacteroides rather than from Escherichia coli, which is a potent innate immune activator. The Bacteroides LPS was found to be structurally distinct from E. coli LPS and inhibits innate immune signaling and endotoxin tolerance. It was observed that unlike LPS from E. coli, Bacteroides dorei LPS does not decrease incidence of autoimmune diabetes in non-obese diabetic mice. Early

colonization by immunologically silencing microbiota may thus preclude aspects of immune

education (170).

Rheumatoid arthritis (RA) is an autoimmune disease where the immune system attacks the

joints leading to swollen and painful joints. The mucosal surfaces are sites of RA initiation.

The common occurrence of periodontal dysbiosis in RA suggests that oral pathogens may

trigger the production of disease-specific autoantibodies and arthritis in susceptible

individuals. Periodontitis is characterized by the presence of citrullinated autoantigens that

are primary immune targets in RA. The citrullinome in periodontitis is similar to the

hypercitrullination observed in the rheumatoid joint, implicating this mucosal site in RA

pathogenesis. Recent studies identified the periodontal pathogen Aggregatibacter actinomycetemcomitans as a bacterial trigger of autoimmunity in RA by inducing hypercitrullination in host neutrophils. The pore-forming toxin leukotoxin A secreted by A. actinomycetemcomitans triggers autoantigen citrullination in the RA joint (171). Zhang et al. (172) reported alterations in the gut, dental or saliva microbiome that distinguished

patients with RA from healthy controls. Individuals with RA had low numbers of

Haemophilus spp., whereas Lactobacillus salivarius was very high in these patients. It has been reported in experiments in mice that inoculation with Bifidobacterium adolescentis exacerbated autoimmune arthritis. B. adolescentis is known to induce Th17 cells in the

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intestine (173). Interestingly, the frequencies of Th17 cells and levels of interleukin-17

strongly correlated with systemic disease activity at both the onset and the progression of

RA (174).

Role of microbiome in obesity

Obesity results from an imbalance of food intake, basal metabolism, digestive tract

microbial composition and energy expenditure (175,176). According to Turnbaugh et al.

(177) the gut microbiome should be considered as a set of genetic factors that together with

host genotype and lifestyle contribute to the pathophysiology of obesity. It is observed that

the intestinal bacteria in obese humans and mice differ from those in lean individuals. Obese

mice microbiota was found to be rich in Firmicutes compared to the lean mice microbiota, which was abundant in Bacteroidetes (177). Strikingly, colonization of germ-free mice with microbiota from obese mice was sufficient to cause a significant increase in total body fat, as

compared to colonization with microbiota from lean mice (177). The obese microbiome has

an increased capacity to harvest energy from the diet, thereby increasing weight gain in the

host (177,178). Colonization of adult germ-free mice with a gut microbial community

harvested from conventionally raised mice increased body fat within 10–14 days, despite an

associated decrease in food consumption. This change involves several linked mechanisms:

microbial fermentation of dietary polysaccharides that cannot be digested by the host;

subsequent intestinal absorption of monosaccharides and short-chain fatty acids; their

conversion to more complex lipids in the liver; and microbial regulation of host genes that

promote deposition of the lipids in adipocytes (179).

Transfer of human microbiota to mice can phenocopy such effects, as shown by Ridaura et

al. (180). Cohabitation of mice harboring an obese microbiota with mice containing the lean

microbiota prevented the development of increased body mass and obesity-associated

metabolic phenotypes in obese cage mates. Rescue correlated with invasion of specific

members of Bacteroidetes from the lean microbiota into obese microbiota and was diet- dependent. The study confirmed that specific bacteria along with diet could induce obesity.

Childhood obesity is considered one of the most serious global health issues in our society.

Obese children are more likely to be obese in adulthood and are at greater risk of premature

death and adverse health outcomes in later life (181). Administration of three or more

courses of antibiotics before children reach age 2 years is associated with an increased risk

of early childhood obesity (182). When given early in life antibiotics that disrupt microbiota

composition, and consequently the metabolic activity of the microbiota, can affect the body

mass of the host by either promoting weight gain or stunting growth (183). The correlation

of antibiotics to obesity has been earlier shown in animal models (184).

Food is broken up into components that tend beneficial microorganisms. Bacterial

fermentation of a diet rich in fibers leads to production of short chain fatty acids (SCFA),

which as noted above includes acetate, propionate, and butyrate in the gut (185,186).

Interestingly, butyrate promotes colonic health and helps prevent cancer (185,187,188). The

consumption of high fat and high calorie foods negatively impacts the beneficial microbes,

which are believed in turn to promote obesity. Notably, obese people have lower

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Bacteroidetes and more Firmicutes in their distal gut than lean subjects and the introduction of low fat and carbohydrate diets increased the proportion of Bacteroidetes (175,189).

Obesity is also known to impair cognition and produces atrophy of brain regions associated

with learning and memory. In animal studies it has been shown that, even before the onset of

diabetes or metabolic syndrome, early stage obesity produced deficits on cognitive tasks that

require the prefrontal cortex. Impaired cognition was associated with synapse loss, including

reduced numbers of dendritic spines and expression of synaptic proteins, as well as

structural alterations in the microglia. Thus, obesity must be considered as a contributing

factor to brain dysfunction mediated through the gut-brain axis (190,191).

It has been demonstrated recently that some bacterial species are beneficial to the host by

preventing obesity. In animal models and humans the abundance of Akkermansia muciniphila is decreased in obese and type 2 diabetic mice (192) and use of the bacterium as a probiotic is beneficial to the host. Interestingly, whole bacterium is not essential to prevent

obesity. Intake of the membrane protein of the bacterium per se could be beneficial to the host. Amuc_1100, a specific protein isolated from the outer membrane of A. muciniphila, interacts with Toll-like receptor 2, is stable at temperatures used for pasteurization, improves

the gut barrier and recapitulates the beneficial effects of the bacterium (193).

Metformin is a well-established drug in the management of type II diabetes and obesity.

Recent studies suggest that the microorganisms are involved in mediating the beneficial

effects of metformin on glucose metabolism. Metformin shifts gut microbiota composition

through the enrichment of mucin-degrading A. muciniphila as well as several short- chain fatty acid-producing microbiota including Butyrivibrio, Bifidobacterium bifidum, Megasphaera, and Prevotella (194). Overall, there is substantial evidence of the key role of microbiota in obesity and its adverse effects.

Microbiome and cardiovascular diseases

The gut microbes produce a large range of metabolites which act not only in the gut, but also

systemically, and this large pool of known and unknown metabolites is not fully understood

(195). The metabolite trimethylamine N-oxide (TMAO) is the first potentially direct link between the gut microbiota and atherosclerotic heart disease. Trimethylamine (TMA) is

produced by the gut microbiota from nutrients containing l-carnitine, choline, and

phosphatidylcholine, and is subsequently oxidized by hepatic flavin-containing

monooxygenases to TMAO. TMAO has been proposed to interfere with cholesterol

transportation, and TMAO precursors promote foam cell formation and atherosclerosis in

animal models, but not in the presence of antibiotics to the drinking water, suggesting a

microbiota-dependent mechanism (195).

Hypertension is a risk factor for coronary heart disease, yet whether gut microbiota

dysbiosis is involved in the development of hypertension remains largely unknown. In a

recent study it was observed that the bacterial genus Prevotella and Klebsiella were overrepresented in individuals with hypertension. Fecal transplantation from hypertensive

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human donors to germ-free mice increased blood pressure in animals thereby demonstrating

the direct influence of gut microbiota on high blood pressure (196).

Beneficial microorganisms are known to protect against atherosclerosis. The lack of gut

microbiota in germ-free apolipoprotein E (ApoE)-null mice, an experimental model of

human atherosclerosis, was found to induce the development of atherosclerotic plaques even

when animals were fed a standard low-cholesterol diet. Colonization with normal human

microbiota prevented atherogenesis in germ-free ApoE-null mice fed a standard low-

cholesterol diet but not a diet with high cholesterol content (197). The bacterial genera

Eubacteria, Anaeroplasma, Roseburia, Oscillospira and Dehalobacteria appeared to be

protective against atherosclerosis and showed significant negative correlation with

atherosclerotic plaque size and plasma adipocyte - fatty acid binding protein (A-FABP) and

cholesterol (198). A. muciniphila is also beneficial to the heart. The bacteria attenuate atherosclerotic lesions by ameliorating metabolic endotoxemia-induced inflammation

through restoration of the gut barrier (199).

Microbiome and behavior

The exponential growth in our collective knowledge of the human microbiome has seen the

study of gut microorganisms move beyond the traditional preserve of strictly

microbiological disciplines. As our appreciation of the structure and dynamics of the gut

microbiome has flourished, so too has our grasp of the implications for host physiology in

health and disease. Perhaps one of the more surprising aspects of this host-microbe dialogue

is the complex interactions that manifest as alterations in brain and behavior. Moreover, the

bidirectional nature of this conversation needs to be considered in the context that

disruptions to CNS function can be expressed distally as alterations in microbiome

composition and function in the gastrointestinal tract. These aspects of host-microbe

dialogue are generally important to medicine, due to the impact of behavioral states that

widely impact and/or reflect the operation and progression of pathogenic processes and their

treatment (e.g., the negative impact of depression on general therapeutic compliance).

These complex reciprocal interactions are facilitated by the microbiome-gut-brain axis,

which incorporates the gut microbiome as a critical node of the communication network

encompassing the CNS, the neuroendocrine and neuroimmune systems, the sympathetic and

parasympathetic arms of the autonomic nervous system and the enteric nervous system

(200). The focus on the gut microbiome has proved to be a surprisingly fertile ground, and

the evidence garnered from a variety of preclinical approaches has converged to illuminate

how the gut microbiome regulates multiple behaviors, physiological readouts and indeed

many fundamental aspects of brain function.

In this regard, surveys of microbiota-deficient germ-free animals have proved particularly

informative. The use of these animals in general is not new, but their application to CNS-

directed queries has been a notable feature of recent research efforts (201,202). From a

behavioral perspective, these animals display a less-anxious phenotype (203-206) and this

atypical performance can be normalized if the animals are colonized post-weaning (205).

Remarkably, it has also been demonstrated using both the germ-free paradigm and an

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antibiotic-induced microbiota deficiency that anxiety-like behaviors can be transferred via

the gut microbiota by means of a fecal transplant (207,208). Germ-free animals also display

alterations in social behaviors (209,210) and, insofar as it has been logistically possible to

address in detail in this paradigm, aspects of cognitive function (211). Gut microbiota

depletion using a cocktail of antibiotics from early adolescence in mice replicates many of

the behavioral characteristics of germ-free mice, including reduced anxiety-like behaviors

and impaired cognitive performance (212).

Other approaches have both largely supported and extended the behavioral picture painted

by microbiota-deficient animals. For example, administration of a probiotic Lactobacillus rhamnosus strain reduced anxiety and depression-related behaviors (213), while alternative candidate probiotics, including a Bifidobacterium longum strain, exerted a beneficial impact on cognitive processes (214). Prebiotics (eg. fiber rich foods that can influence the

microbiome) can also exert anxiolytic effects (215), while bacterial infection with an enteric

pathogen can impact both learning and memory (211) and modulate pain behaviors (216).

Physiologically, germ-free animals also exhibit profound differences with conventionally

colonized controls. These differences include a defective immune system and exaggerated

corticosterone outputs to acute stressors (205,217,218). The microbiome is also required for

the development of microglia, cells that defend the central nervous system. Microglia from

germ-free mice had altered gene expression that influenced its development (219).

An increased availability of tryptophan - the amino acid precursor to neuroactives such as

serotonin and kynurenine pathway catabolites, as generated respectively by IDO1 or TPH -

is one feature of the germ-free state (205,220,221). Many aberrant physiological features can

be rescued if the animals are colonized with a normal microbiota, especially if this

intervention takes place during specific time windows post weaning (205,217). As is the

case for behavior, other microbiota manipulations such as rendering mice microbiota

deficient or probiotic ingestion by rodents can also impact parameters such as corticosterone

outputs or tryptophan availability (212,213,222).

The reciprocal interaction between stress and the microbiome is a particularly interesting

facet of this bidirectional relationship. As indicated above, the gut microbiota exert an

influence on the hypothalamic-pituitary adrenal axis, the main host stress response system

and this can be captured by measuring cortisol in humans or corticosterone in rodents (223).

There are now studies showing that the opposite is also true and that a variety of stressful

insults which are linked to psychopathology in adulthood can alter the composition of the

gut microbiome in animal models. This is reflected in studies that have examined early-life

stress (224,225), prenatal stress (226,227) and psychosocial stress (228-230). Interestingly,

the gut microbiota seems necessary for the expression of some of the pathological

behavioral features induced by maternal separation (231), a well validated early life stress

based model of gut-brain axis dysfunction (232). In the clinical setting, maternal prenatal

stress is also associated with alterations in the infant gut microbiome (233). Another

example of such feedback loop is stress-related microbiome-gut-brain axis dysfunction in

irritable bowel syndrome (234).

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Growing up, germ-free influence biological function such as blood-brain barrier integrity

(235), transcriptional regulation (203,236), neurogenesis (237) microglial function (238) and

myelination (239). Recently, it has been demonstrated that a germ-free mouse model of

Alzheimer's disease displayed a marked reduction of cerebral amyloid pathology and that

colonization of these mice with the gut microbiota of their conventionally-colonised

counterparts reinstated the amyloid pathology (240). Although the behavioral implications

of these altered amyloid phenotypes requires elaboration, this intriguing study does provide

support for the hypothesized role of the gut microbiota in neurodegenerative disorders (241).

The mechanisms underpinning influence of the gut microbiome on brain and behavior are

under investigation. The gut microbiota are required for motor deficits, microglia activation,

and α-synuclein pathology. Colonization of microbiota from Parkinson's disease-affected patients enhances physical impairments compared to microbiota transplants from healthy

human donors. Thus the gut bacteria is involved in movement disorders and alterations in the

human microbiome represent a risk factor for Parkinson's disease (242). Recently the role of

the vagus nerve (the main neural communication highway between the gut and the brain) has

attracted much attention (243). It has been demonstrated, for example, that the beneficial

CNS impact of a probiotic was abolished in vagotomised mice (213) while the anxiety-like

behaviors that emerge in a colitis model were absent in previously vagotomised mice (207).

The vagus nerve is not the sole conduit linking the gut and the brain (244) and a variety of

alternatives have been considered. These include microbial regulation of tryptophan

metabolism (245), microbial metabolites such as short chain fatty acids (SCFA) (246) or

indoles derived from tryptophan (247), neuropeptide production (248) as well as

immunomodulation (249). The important role of the gut microbiota in maintaining intestinal

barrier integrity also needs to be taken in account (250,251).

The landscape for manipulating the microbiome is broad and increasingly financed (252).

Consideration are being given to priming interventions which promote assembly of the

infant microbiome (65,253,254), sustain the gut microbiota in healthy aging (125,126), more

radical options such as fecal microbiota transplantation (255) as well as less controversial

options such as psychobiotics (256), exercise (257,258) and diet-based manipulations

(259,260). Indeed, a number of small studies using healthy volunteers have now

demonstrated that ingestion of certain cocktails of probiotics, a fermented milk product with

probiotic or prebiotics can impact on the CNS (261-264). Autism Spectrum Disorders

(ASD) are complex neurobiological disorders characterized by impairment in social

interaction and communication and restricted, repetitive, and stereotyped patterns of

behavior, interests, and activities. Autistic children suffer from gastrointestinal disorders.

Autistic children have less diverse microbial population in the gut and significantly lower

abundances of the genera Prevotella, Coprococcus, and Veillonellaceae involved in carbohydrate metabolism (265).

Future directions will likely see further elaboration of the role of the gut microbiome in

sleep (266) and circadian rhythms (267-269). Our awareness of the interface between natural

and built environments, the gut microbiota and human behavior is also growing (270-272).

Of course, a key caveat is to what degree this promising but largely preclinical body of

research will effectively impact the clinical setting. Moving from mouse to man, be it in the

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context of CNS-directed or gastrointestinal-focused research, is of course complicated for

stress-related neuropsychiatric and other heterogeneous disorders associated with the gut

microbiome (273,274). Of equal importance is the necessity to address the issue of whether

the correlations that have been noted thus far between multiple disorders and the gut

microbiota alterations are in fact causal relationships. Such obstacles are not insurmountable

with due diligence and the necessary multidisciplinary expertise required to exploit the

considerable opportunities presented by host-microbe interactions.

Beneficial microorganisms restrict the outgrowth of pathogens in the gut

The human microbiota encompasses all the microorganisms that reside on the skin and in all

other tissues and organs including the gastrointestinal tracts. Of these body sites, the

gastrointestinal (GI) tract is the most densely colonized organ. The microbiome includes

bacteria, fungi, and archaea (275). There are approximately 1000 species of microbes

colonizing the gut, with densities of 104 to 105 bacteria per millimeter of digestive effluent

in the proximal small intestine and 1011 bacteria per gram of luminal content in the colon

(276). The physicochemical conditions in the gut influence the composition of the intestinal

microbiota (277). The GI tract harbors many distinct niches, each containing a different

microbial ecosystem that varies according to the location within the GI tract. The microbial

density increases along the GI tract with101–104 microbial cells in the stomach and

duodenum, 104 to 108 cells in the jejunum and ileum, to 1010 to 1012 cells per gram in the

colon and feces (277-279).

The majority of all microorganisms in the human digestive tract are bacteria and belong to

two phyla, the Bacteroidetes and the Firmicutes (280). In addition, the other significant phyla occupying the digestive tract includes Proteobacteria, Actinobacteria, Fusobacteria, Spirochaetes, Verrucomicrobia and Lentisphaerae (281,282). The methanogens, Methanobrevibacter and Methanosphaera are the most dominant archaeal groups (7,283). The two common fungal phyla in the gut include Ascomycota (which includes the genera Candida and Saccharomyces) and Basidiomycota (30,284).

Intestinal microbiota play a central role in the metabolic, nutritional, physiological and

immunological processes of the human body, processing indigestible dietary

polysaccharides including resistant starch and dietary fibers thereby leading to the

production of important nutrients, such as SCFAs, vitamins (vitamin K, vitamin B12, folic

acid) and certain amino acids that humans are unable to synthesize themselves

(279,285,286). The plant polysaccharides in our diet are rich in xylan-, pectin-, and

arabinose-containing carbohydrate structures. The human genome lacks most of the

enzymes required for degrading these glycans. Nevertheless, the distal gut microbiome

provides us with this capacity to process these polysaccharides. The human gut microbiome

is enriched for genes involved in glucose, galactose, fructose, arabinose, mannose, and

xylose, starch and sucrose metabolism. Our microbiome also has significantly enriched

metabolism of glycans, amino acids, and xenobiotics; methanogenesis; and 2-methyl-d-

erythritol 4-phosphate pathway-mediated biosynthesis of vitamins and isoprenoids (7). The

intestinal microbiota also participates in the defense against pathogens by mechanisms such

as colonization resistance and production of antimicrobial compounds. Furthermore, the

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intestinal microbiota is involved in the development, maturation and maintenance of the GI

sensory and motoric functions, the intestinal barrier and the mucosal immune system (279).

The microbiota of the intestine is also involved in promoting bone formation as well as

resorption leading to skeletal growth. Microbiota induces the hormone insulin-like growth

factor 1 (IGF-1), promoting bone growth and remodeling. When the microbiota ferment

fiber short-chain fatty acids (SCFAs) are produced leading to induction of IGF-1 that

promote bone growth (287).

The very high microbial content of the large intestine poses a major challenge to the

mucosal immune system, as it needs to tolerate commensal microbiota and dietary antigens

while maintaining the ability to eliminate pathogens. Induction of colonic Treg is crucial in

fostering this immune homeostasis (288). CD4+CD25+FOXP3+ Treg are of two types:

thymus-derived Treg (tTreg) and peripherally derived Treg (pTreg). Although it is difficult to

distinguish these types phenotypically, both are thought to have an essential role in immune

regulation (288). While tTreg develop in the thymus, the major site for pTreg development is

the colon, resulting in a large population of regulatory cells that have a distinct TCR

repertoire and are critical for intestinal homeostasis (152). Notably, the development of

pTreg requires microbiota to be present in the colon. Though the mechanism of induction of

colonic pTreg is not understood, several microbial components have been found to enhance

their expansion and function, including SCFAs and polysaccharide A of Bacteroides fragilis

(288). Acetate, propionate, and butyrate are the three main SCFAs, and butyrate has been

found to be the most potent inducer of colonic Treg.

The newborn infant is colonized at birth with microbes from the mother's vaginal and fecal

microbiota as well as with other environmental microbes encountered in the first days of life

(289). The mode of delivery influences the microbial composition in man. A paper by

Penders et al. (93) demonstrated that the important determinants of the gut microbiome

composition in infants were the mode of delivery, type of infant feeding, gestational age,

infant hospitalization and antibiotic use in the infant. Term infants born vaginally and

exclusively breastfed had the most “beneficial” gut microbiota (had the highest numbers of

Bifidobacteria and lowest numbers of C. difficile and E. coli). In contrast, infants born through C-section had lower numbers of Bifidobacteria and Bacteroides, and they were more often colonized with C. difficile, as compared to vaginally born infants. Exclusively formula-fed infants were more often colonized with E. coli, C. difficile, Bacteroides and Lactobacilli, as compared with breastfed infants. Hospitalization and prematurity were associated with higher prevalence and counts of C. difficile. Administration of antibiotics to infants was associated with decreased numbers of Bifidobacteria and Bacteroides. Infants with older siblings had slightly higher numbers of Bifidobacteria, compared with infants without siblings (93,290).

Clostridium difficile is an opportunistic, anaerobic gram-positive, spore-forming, toxin- producing bacillus that is transmitted among humans through the fecal–oral route. Notably,

the pathogen is generally present in the human gut but it does not cause any disease under

normal conditions. Abuse/misuse of antibiotics destroys beneficial microbiota that enables

the proliferation of C. difficile leading to pathogenic conditions. Ampicillin, amoxicillin, cephalosporins, clindamycin, and fluoroquinolones are the antibiotics most frequently

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associated with disease, but almost all antibiotics have been associated with increased rates

of opportunistic infection. C. difficile colonizes the large intestine and releases the exotoxins TcdA and TcdB that cause colitis in susceptible persons (291,292). C. difficile has emerged as a major enteric pathogen with worldwide distribution. In the United States, C. difficile is the most frequently reported nosocomial (i.e., hospital-acquired) pathogen. A recent

surveillance study identified 453,000 cases of C. difficile infection and 29,000 deaths associated with C. difficile infection; approximately a quarter of those infections were community-acquired (293). The antibiotics prescribed to control C. difficile include metronidazole, vancomycin, fidaxomicin or surgery in extreme cases of infection (294).

Fecal transplant is emerging as an alternative strategy for treating recurrent C. difficile infections (295). Use of probiotics (such as beneficial bacteria and yeast), which help restore

a healthy balance to the intestinal tract are safe and effective for preventing C. difficile- associated diarrhea (296). Thus, beneficial microorganisms are essential to maintain the

human gut immune homeostasis thereby preventing pathogenic infections. Further

contributing to such defenses, beneficial microorganisms also modulate epithelial cell

proliferation, villus architecture and angiogenesis within the intestine, along with xenobiotic

metabolism, bone mineral density, behavior and key metabolic functions (297,298). Tipping

the balance favoring the expansion of enterobacteria is one of the causes of several

inflammatory bowel diseases. However, it is not known how the favorable bacteria prevent

dysbiosis. A recent study demonstrated that microcins, the small proteins secreted by several

favorable bacteria could limit the expansion of competing Enterobacteriaceae (299).

Microbiome effects on intestinal barrier function and inflammatory bowel

disease

Mammals, including humans, support one of the most complex microbial ecosystems.

Although the immune system is classically thought to have evolved to provide protection

from infection by microbial pathogens, animals peacefully coexist with a vast and complex

microbiota, which extensively interacts with the immune system. It has recently been

proposed that the total information encoded by the mammalian genome is not sufficient to

carry out all functions that are required to maintain health and that products of our

microbiome are crucial for protection from various diseases (300). It is possible that

alterations in the development or composition of the microbiota (dysbiosis) disturb the

partnership between the microbiota and the human immune system, ultimately leading to

altered immune responses that may underlie various human inflammatory disorders (146).

In inflammatory bowel disease (IBD), the role and interplay of the microbiome (301-303)

with a GI barrier compromise (304-306) has been the subject of extensive review (307-311).

GI barrier function is not governed solely by the tight junctional (TJ) complex, although this

focus has certainly attracted the greatest basic research interest. TJs form the continuous

intercellular barrier between epithelial cells, which is required to separate tissue spaces and

regulate selective movement of solutes across the epithelium (312). From a wider

perspective, GI barrier function also may be compromised by an impaired mucus layer over

the epithelium (a topic reviewed nicely by others recently [Chen et al., (313)]), as well as by

cell death in the epithelium or an epithelial-mesenchymal transition leading to impaired cell

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adhesion/attachment (to other cells and substratum). Leak at sites of compromised TJ may

be quite distinct in nature from leak at sites of cell death (314). Likewise the remediation of

leak is very different in these cases – repair of leak from impaired TJ may be a purely

transcription/translation/phosphorylation-based process, whereas remediation of leak due to

cell death/dedifferentiation/detachment could also require a careful orchestration of cell

motility and cell replication. In these different cases, the microbiome may exert control over

very different processes. It is worth considering that any given specific case of IBD likely

involves gastrointestinal leak from all of these causes, and therefore alleviation of such leak – and the inflammatory cascades it gives rise to – is a quite complex task. Further research is

required to determine whether microbiome may be better at repair of one type of leak than

the other.

There is no doubt that IBD is in part driven by a breakdown or compromise of the

gastrointestinal epithelial barrier, and many reviews on IBD have dealt with this feature as

mentioned above. There is some controversy concerning whether a compromise of barrier

function is the initial causation of the disease. The findings that asymptomatic, first degree

relatives of IBD patients in fact harbor molecular-level leak in their GI tract mucosa has

traditionally been powerful evidence tilting the argument toward causality (315,316). The

question of course that leaps to the fore then is what induces the leak in the first place? The

very fact that a genetic element exists in IBD (e.g. in first degree relatives) indicates a role

for genetics in the disease, but equally obvious is that genetics is probably a necessary but

insufficient condition.

Studies focusing on this involvement of gastrointestinal microbiome in IBD take two forms:

1) whether the microbiome is abnormal in IBD and possibly playing a role in etiology; 2)

whether a microbiome modification can be designed as a therapeutic option in the disease.

The second possibility appears achievable: some of the best clinical evidence – on the basis

of its very applicability – is the success of “fecal-transplant” procedures in achieving

therapeutic efficacy in IBD (317,318). Prior to the recent advent of these protocols there was

the use of butyrate enemas to achieve therapeutic relief (286,319,320). The therapeutic

efficacy of luminal administration of butyrate is cogent testimony to the positive role played

by the normal microbiome in maintaining a functional epithelial barrier in the GI tract, as

well as to the utility of targeting the microbiome as a viable clinical approach to IBD.

Butyrate is a significant metabolite of dietary fiber by the normal gastrointestinal microbiota

(321), with butyrate levels in the GI lumen being the highest in the body. Butyrate has been

found in many recent in vitro studies to be highly effective in inducing structural changes to the epithelial TJ complex resulting in improved epithelial barrier function (322,323). A

similar literature also exists for the GI microbiota metabolite, indole, a product of tryptophan

metabolism by commensal bacteria (324,325). In combination, the fecal transplant and

butyrate enema clinical studies, along with the in vitro studies of TJ modification and

enhancement by butyrate and indole, provide a very powerful argument of not only

maintenance and modification of the GI barrier by the microbiota, but also for targeting the

microbiota as a viable, effective therapeutic strategy.

Better delineated proof of the ability of the microbiota to both positively and negatively

affect the GI barrier comes out of animal model and epithelial cell culture studies. For

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example, the probiotic bacterium, Lactobacillus casei, both strengthened barrier function and decreased proinflammatory cytokine content in BALB/c mice (326). In addition, such

treatment also modified the GI microbiota overall. The probiotic and commensal bacteria

Lactobacillus rhamnosus and Faecalibacterium prausnitzii have also improved barrier function, as demonstrated in studies with CACO-2 cell culture models and experimentally

induced colitis in C57BL/6 mice (327,328). The microbiome is also known to be involved in

the wound healing of the mucosa of the gut (329). In mice it has been demonstrated that

mucosal injury leads to increase in the expression of formyl peptide receptor 1 (FPR1) and

neutrophilic NADPH oxidase (NOX2) that causes depletion of oxygen resulting in the

enrichment of anaerobic bacteria. The anaerobic, mucinophilic gut symbiont, Akkermansia muciniphila, stimulated proliferation and migration of enterocytes adjacent to the colonic wounds mediated through FPR1 and intestinal epithelial-cell-specific, NOX1-dependent

redox signaling, thereby leading to wound healing of the mucosa. These findings highlight a

very important consideration in studies of the microbiota, barrier function and IBD – namely

that not only can microbiota affect barrier function, but modification of barrier function

(good and bad) may well affect microbiota composition – a “research road less traveled.”

This less investigated area is well illustrated by the finding that anti-TNF immunologic

medications – which reduce proinflammatory cytokines and allow for barrier repair –also

result in changes in microbiota composition (330).

It is worth noting too that a factor as omnipresent as diet can affect both barrier function and

microbiota, while also simultaneously affecting cytokine production. Administering a high-

fat “Western” diet to CEABAC10 mice induced deleterious changes in GI microbiota (e.g.

increased content of adherent-invasive E. coli), decreased mucus layer protection, and led to GI barrier compromise (331). Pathogenic E. coli have been implicated in Crohn's disease, in part due to an ability to not produce cell death while inducing synthesis of copious amounts

of TNF (332). The dietary connection to an altered microbiota in terms of pathogenic

bacteria is also apparent in the finding that vitamin D deficiency enables the barrier

disruptive effects of pathogenic E. coli to be manifested (333). Even more fascinating and less intuitive is an effect of the environment at large on barrier function, microbiota and

inflammatory status, as Kish et al. (334) show for particulate air pollutants. It will be

instructive to discover in future research whether the principal actions of diet/nutrition/

environment are on microbiota directly and barrier function indirectly, or vice versa.

As more and more studies reveal the intricate interplay between GI barrier function, GI

microbiota and degree of inflammation, it is worth considering that the effects of microbiota

on barrier function - and barrier function on microbiota - will in large degree derive from

actions of protein kinases in signaling pathway transduction systems. This has been recently

very well reviewed by Yang and Yan (335). One would caution however against taking an

aggressively reductionist approach in dealing with the interplay of microbiota, barrier

function and inflammation: The complexity of the gut microbiome in the GI tract, the

complexity of the signaling pathways known to regulate barrier function, and the complexity

of cytokine interactions all suggest strongly that one needs to tread carefully in what may

well be overly ambitious undertakings to find and utilize specific molecular mechanisms

involved in this intricate relationship. A properly functioning gastrointestinal mucosa is in

essence a symphony scored by microbiota “strings,” barrier function “woodwinds,” and

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immune regulatory “brass.” To search for an all-pivotal kinase or phosphatase responsible

for GI mucosal homeostasis may be analogous to trying to claim a single flute or viola as the

fulcrum of Beethoven's Ninth – a fool's errand that ignores the extreme complexity and

subtlety of the GI environment.

Because of the still singular importance of the studies showing epithelial barrier compromise

in asymptomatic first-degree relatives of IBD patients to the field of IBD research, it will be

very interesting to observe the outcome of studies yet to be performed on the microbiome of

first degree relatives of IBD patients. It is likely that those results could be just as pivotal to

the future understanding of IBD as were the now long ago studies of May and Hollander on

barrier function and IBD (315,316).

Increased risk of developing IBD may be due to improved hygiene practices. Ramanan et al. (336) showed that intestinal helminth infection, caused by parasitic worms, protects IBD-

susceptible mice from developing the disease. The infection of parasitic worms increased

specific protective species and limited other inflammatory members of the microbiota.

People from helminth-endemic regions harbored a similar protective microbiota, and their

de-worming led to an increase in inflammatory Bacteroidales, as observed in the mice. Thus, a changing microbial environment may shape susceptibility to inflammatory disease.

Though we currently know at least a partial membership of the human microbiome, we are

yet to fully understand how these microorganisms are contained in the intestine. Anatomy of

the colon by light microscope reveals a mucus layer, mucosa, submucosa, gut associated

lymphoid follicle and muscularis. The intestinal/colonic 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 (337). Colonic mucus

is produced by the goblet cell. The main mucus component in the intestine is MUC2 mucin,

a large and heavily O-glycosylated gel-forming mucin that forms enormous polymeric nets

by C-terminal dimerization and N-terminal trimerization. Upon secretion from the goblet

cells, the mucus expands rapidly and builds a stratified dense layer that is attached to the

epithelium. Normal human sigmoid colon has an inner mucus layer that is impenetrable to

bacteria. At a distance, far from the epithelial surface, the inner mucus is transformed into a

soluble and less organized outer mucus layer that by proteolytic expansion, generates the

preferred habitat for the commensal microbes (338).

IBDs are characterized by aberrant innate and adaptive immune responses to commensal

luminal bacteria (339). Ulcerative colitis (UC) is thought to be caused by some strains of E. coli (340). In cell culture models it has been showed that UC-associated E. coli producing α- hemolysin can cause rapid loss of TJ integrity (341). The human intestinal epithelium is

formed by a single layer of epithelial cells that separates the intestinal lumen from the

underlying lamina propria and the space between these cells is sealed by TJ, which regulate

the permeability of the intestinal barrier (342). TJ complexes allow passive absorption of

small hydrophilic molecules (nutrients and ions), but they restrict passage of large molecules

and infectious microbes. UC is characterized by a leaky intestinal barrier due to defective

TJ. Our group had reported that attenuation of the Bin1 gene in a mouse model would protect against experimental colitis (343). Based on the study we recently demonstrated that

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treatment of experimental colitis with Bin1 monoclonal antibody would support mucosal

barrier function by inducing the expression of TJ proteins thereby protecting the integrity of

the lymphoid follicle. The therapy may be a novel strategy to treat UC and possibly limit

risks of colorectal cancer (344). Thus lowering Bin1 levels may be a strategy that would lead

to enhanced TJ proteins that, in turn, protects against pathogenic microorganisms from

crossing the epithelial cells.

Other strategies may be used by the epithelial cells to protect against pathogenic microbes

from entering the tissues. Recently while working on subconfluent CACO-2 cells (derived

from the intestine) we observed that EEA1 endosomes (early endosome marker) were

confined more toward the periphery of CACO-2 cell monolayers. To confirm whether these

endosomes are present in the colon tissues, we stained for EEA1 in mouse colonic tissues.

We observed EEA1 endosomes in the peripheral mucosa as well as in the muscularis. Insofar

as endosomes are traditionally thought to protect against foreign bodies including

microorganisms, one might speculate that early endosomes help protect against bacteria

crossing the mucus. Microbes that overcome this barrier to cross the mucus layer might be

destroyed within endosomes, with further protection afforded by the endosomes lining the

muscularis (Fig. 1).

Additional endosomal strategies may be used to protect colonic tissues against the entry of

pathogenic microbes. Endosomes are traditionally thought to protect colonic epithelial cell

layers against microorganisms, possibly helping eliminate bacteria that cross the mucus

layer, a strategy that also may be extended into the subordinate muscularis layer (Fig. 1). In

subconfluent human colonic cells, which form island-like colonies in monolayer culture, the

early endosome marker EEA1 can be seen to preferentially localize to the colony periphery.

Similarly, in colon tissues early endosomes display the same localization with EEA1

staining in the periphery of the mucosa and muscularis. Beclin1-dependent autophagy

associated with the endosome pathway also has been implicated in the bacterial and viral

pathogen elimination (345), which downregulate Beclin1 to promote virulence and infection.

Beclin1 associates with endosomes and regulates EEA1/early endosome localization and

late endosome formation (346). Upon TLR signaling, Beclin1 rapidly translocates to the

phagosome and mediates efficient phagosome-lysosome fusion to ensure rapid acidification

and efficient destruction of the pathogen (345). In human colonic cells, Beclin1 staining

occurs throughout the colon tissue at endosomes (Fig. 2), possibly helping direct pathogenic

cargo to lysosomes and thereby restricting microbiome ecology to the gut lumen.

Indoleamine 2, 3-dioxygenase (IDO1) and the microbiome-host interaction

As noted above, establishing and maintaining the symbiotic mutualism that exists between

the microbiome and its mammalian host necessitates the engagement of mechanisms of

acquired immune tolerance as these microorganisms represent the epitome of non-self.

Indoleamine 2,3-dioxygenase-1 (IDO1) is a metabolic enzyme that has gained recognition as

an important mediator of acquired immune tolerance. IDO1 catalyzes the rate-limiting first

step in the degradation of the essential amino acid tryptophan, but is not involved in

maintaining tryptophan homeostasis, which instead is the role of the distinct liver enzyme

TDO2 (tryptophan dioxygenase) (347). The concept of IDO1 as an immune regulator

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emerged from findings that tryptophan catabolism could suppress cytotoxic T cell activation

(348,349). The demonstration that the IDO pathway inhibitor 1-methyl-tryptophan (1MT)

could elicit T cell-dependent rejection of allogeneic mouse concepti (350,351) established

the physiological relevance of tryptophan catabolism as a mediator of acquired immune

tolerance. Subsequent findings linking attenuation of the tumor suppressor gene Bin1 to IDO1 dysregulation and tumoral immune escape (352) provided experimental substantiation

for the corollary proposition that tumor cells might, by inducing IDO1, appropriate this

mechanism of protection for the ‘foreign’ fetus to overcome immunosurveillance. Within the

complex inflammatory milieu of the tumor microenvironment, IDO1 induction is not

necessarily restricted to tumor cells, and non-malignant stromal cells expressing IDO1 can

promote tumoral immune escape as well (353). In particular, IDO1 induction in antigen-

presenting cells, such as dendritic cells and macrophages, has been implicated in promoting

immune tolerance by suppressing effector cytotoxic T lymphocytes (CTL), converting naïve

T lymphocytes to FoxP3+ Treg cells and elevating the suppressive activity of “natural” Treg

(354).

A great deal of attention is now focused on the therapeutic potential of small molecule

inhibitors of IDO1 for treating cancer patients (355), particularly in combination with cancer

chemotherapy or “immune checkpoint” antibodies (352,356). It is not yet clear how IDO1

may influence host interactions with the microbiome, but there has been much attention to

the related topic of its role in the host response to infection by various pathogens, which has

been a topic of interest for a number of years. Indeed, well prior to findings of its

involvement in immune modulation, it was noted that intraperitoneal administration of

bacterial LPS could induce IDO1 activity in the lungs of mice by 30- to 50-fold (357). This

initial indication that IDO1 induction might be associated with the inflammatory response to

microbial infection was followed by reports of pulmonary IDO1 induction in response to

virus infection (358) and IFNγ (359).

Since IFNγ plays a major role in controlling a variety of infections, the finding that IDO1 is highly responsive to IFNγ spurred investigations addressing whether IDO1 might have a downstream anti-microbial effector role. In 1984, Pfefferkorn and colleagues reported that

tryptophan degradation was responsible for the IFNγ-mediated restriction of the growth of the obligate intracellular protozoan Toxoplasma gondii in human fibroblasts (359). IFNγ- mediated restriction of the growth of the obligate, intracellular, gram-negative bacterium

Chlamydophila psittaci was likewise linked to tryptophan deprivation (360). These studies focused attention on IFNγ- elicited tryptophan deprivation resulting from the induction of IDO1 as mediating the anti-proliferative effect on these intracellular pathogens. This

assessment, that IDO1 provides a beneficial effect in combating infections, was complicated

by additional studies demonstrating the activation of genes for Chlamydia persistence triggered by IDO1 mediated tryptophan depletion. Because the bacterium is sensitive to the

antibiotic treatment only when it is metabolically active, IDO1 activity in this context was

detrimental to clearing infections leading to the suggestion that tryptophan supplementation

might help overcome antibiotic resistance (361). Furthermore, while the effects of IDO1 on

microorganisms were initially attributed to depletion of tryptophan, evidence of microbial

effects produced by downstream tryptophan metabolites, from what is collectively referred

to as the kynurenine pathway, were also reported (362,363). In particular, 3-

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hydroxykynurenine was shown to suppress the proliferation of Staphylococcus aureus in vascular allografts (364), and both 3-hydroxykynurenine and 3-hydroxyanthranilic acid were

found to contribute to controlling the replication of Trypanosoma cruzi in mice (365). In a single report, treatment with 1MT produced three different outcomes depending on the

nature of the infection, exacerbating T. gondii toxoplasmosis, restraining L. major leishmaniasis, and having no apparent effect on HSV-1 replication or latency (366). In

aggregate, the implication from these studies of infectious pathogens is that the overall

impact of IDO1 activity, both in terms of tryptophan depletion and the production of various

metabolites, on the diverse ecology of the commensal microbiome is likely to be complex

and contextual.

Perhaps even more consequential than the direct effects of IDO1 activity on particular

microbes are the effects that IDO1 can exert on the overall inflammatory environment. In

accord with its ability to elicit T cell suppression, the general assumption has been that

IDO1 should act in an immunosuppressive manner to limit the severity of inflammation.

Data supporting this interpretation have been reported in a mouse model of chronic

granulomatous disease in which defective IDO1 function in mice lacking an essential

component of NADPH oxidase, p47phox, was implicated in the exaggerated inflammatory

response to infection with Aspergillus fumigatus (367). The more severe illness was associated with higher numbers of IL17-producing γδT cells and fewer IL10 producing αβTreg, which could be reversed by the provision of exogenous kynurenine. However, as with the effects of tryptophan catabolism on microorganisms, the categorization of IDO1 as

strictly immunosuppressive may be an oversimplification (368). In a mouse model of

chemical carcinogenesis, genetic loss of IDO1 did not exacerbate inflammation in response

to phorbol ester-elicited tumor promotion, as would be expected if it were broadly

immunosuppressive, but did result in a dramatically reduced incidence of premalignant

lesions (369). Perhaps even more strikingly, in a mouse model of rheumatoid arthritis (RA),

1MT treatment suppressed rather than exacerbated joint inflammation (370), while in a

contact hypersensitivity model, genetic loss of IDO1 resulted in diminished ear swelling

(371). Why IDO1 has such varied effects on the inflammatory response remains to be fully

elucidated, but suggests that the outcome of the interactions with the complex microbiome

may be contextual and difficult to predict.

The study in the RA model noted above also highlights a particular complication with

interpreting results of the many studies that have relied on the use of the compound 1MT to

inhibit IDO1 activity. Biochemical and pharmacological evaluation of this compound clearly

indicates that it is not directly inhibiting the enzyme at the dose ranges administered in vivo,

and it is able to signal as a mimetic for tryptophan sufficiency and thereby interfere with

activation of downstream response pathways (372). However, a tryptophan deficiency signal

can be provided by any of the enzymes that catalyze tryptophan degradation (IDO1, IDO2,

TDO2 as well as TPH, the latter of which initiates an alternate pathway of tryptophan

catabolism to serotonin). Therefore, 1MT is not a valid tool to discriminate which of these

particular enzymes is involved. Indeed, in the RA model, genetic analysis revealed that the

recently identified paralog IDO2, and not IDO1, is likely to be responsible for the effect of

1MT on joint inflammation, as the effect of 1MT administration was phenocopied in mice

lacking IDO2 but not IDO1 (370,373). Studies using later generation, direct enzyme

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inhibitors (355), coupled with studies in genetically modified animals (371,374,375) can

overcome ambiguities in data interpretation associated with 1MT treatment. Indeed, a recent

study of LPS responses utilizing the IDO1, IDO2 and Tdo2 gene deletion mouse strains provides confirmatory evidence that these three genes have distinct, non-overlapping roles in

the host immune response to this microbial signal (376).

While the regional microbiome present at all barrier surfaces is likely to influence immunity

locally, the microbiome of the gastrointestinal tract is of particular interest because of the

broader role it has been found to play in shaping systemic immune homeostasis (377).

Correspondingly, current microbiome research is largely focused on the gastrointestinal

tract, where commensal microorganisms have been found to contribute to host defense by

limiting the growth of enteric pathogens and producing symbiosis factors that control

intestinal inflammation and pathology (377). Evidence implicating IDO1 in this process has

come from a study of the protective capacity of Lactobacillus salivarius, which is abolished in mice lacking the gene encoding NOD2, an intracellular pattern-recognition molecule that

regulates inflammatory pathways in response to detection of bacterial peptidoglycans. In this

model, IDO1 upregulation was found to correlate with NOD2-dependent protection (378).

The increased regulatory complexity imposed by the gut microbiome may help also explain

counterintuitive findings associated with IDO1 in this tissue. In a dextran sodium sulfate/1,2-

dimethylhydrazine (DSS/DMH) elicited model of colon carcinogenesis, genetic loss of

IDO1 resulted in increased tumor frequency (379), unlike other organ systems in which

IDO1 loss has been associated with resistance to carcinoma development (380,381). This

outcome is similar to the atypical impact on tumor formation ascribed to Treg in the gut,

where their presence appears to be protective against carcinogenesis (382), despite evidence

that Treg are generally associated in other organs with tumor promotion. It has been

proposed that the effect of immunosuppressive mechanisms on inflammatory pathology of

the gut may be quite different depending on whether there is any initial involvement of tissue

damage, as the resulting microbial translocation produces a severe tumor-promoting

inflammation (383). Under these circumstances, dampening the inflammatory response via

immune suppressive mechanisms may provide a more consequential benefit that

overshadows any detrimental role in promoting immune escape. This interpretation is

consistent with the findings of two otherwise apparently contradictory studies in colitis

models, where IDO1 blockade resulted in augmented colitis induced by trinitrobenzene

sulfonic acid (384), but diminished colitis induced by Citrobacter rodentium (385).

In conjunction with the complex immune regulatory effects attributed to IDO1 activity in the

gut, tryptophan metabolites produced by the microbiota affect mucosal reactivity. When

switching from sugar to tryptophan as an energy source, the highly adaptive lactobacilli in

the gut expand and produce an aryl hydrocarbon receptor (AhR) ligand, indole-3-aldehyde,

that contributes to AhR-dependent IL-22 transcription. The resulting IL-22-dependent

mucosal response promotes the survival of mixed microbial communities, while providing

colonization resistance to the fungus Candida albicans and mucosal protection from inflammation. This example of coevolutionary commensalism through the microbiota-AhR

axis represents yet another way in which tryptophan catabolism appears to be involved in

fine tuning host mucosal reactivity (386). As further investigations into the physiological and

pathophysiological interactions between IDO1, the commensal microbiome and host

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immunity are conducted, the indications from these early studies are that IDO1 is likely to

play an integral but contextual role at the interface between homeostasis and dysbiosis.

Microbiome and cancer

The interplay between microbes, cancer and the immune system is in no manner fully

defined. However, accumulating evidence argues provocatively that microbes exert a variety

of functions on host oncogenesis, tumor progression and response to immunotherapy. Thus,

selectively manipulating the gut microbiome is a critical parameter to consider in the

ongoing battle against established cancers.

The metabolic potential of the gut microbiota is now regarded as vital to the process of

malignant transformation. Disruption of the intimate relationship between the host and

intestinal bacteria, known as dysbiosis, can affect oncogenesis, tumor progression, and

response to cancer therapy. Dysbiosis can occur for several reasons: 1) direct occupancy of

unwanted, foreign microbes (as discussed above) that outcompete friendly gut flora, 2) a

response to immunosenescence with aging, and 3) direct environmental insults such as

antibiotics and smoking (387). In the setting of chronic autoimmune processes such as

Crohn's disease and UC, the integrity of gut epithelial, myeloid, and lymphoid components

are disrupted (Fig. 3). These chronic insults ultimately increase host's risk for neoplastic

transformation (388). Indeed, several factors that favor carcinogenesis similarly recapitulate

dysbiosis.

One well-studied model of the dysbiosis/cancer connection is that of repeated intra-

abdominal infections, the use of antibiotics, or both leading to an increased incidence of

colorectal cancer (389). In several preclinical studies, interventions that abrogate or directly

alter gut microbiome composition increase the incidence and progression of colorectal

carcinoma in both genetic and carcinogen-induced models of tumorigenesis (390-392).

Moreover, several by-products of the gut microbiota directly target intestinal epithelial cells

and either facilitate oncogenesis (as reported for hydrogen sulfide and the Bacteroides fragilis toxin) or suppress tumorigenesis (in the case of SCFAs) (393). Intestinal microbes have been characterized to participate in more than just colorectal carcinogenesis.

Experimental models of gut flora also elucidate the development of other extraintestinal

cancers such as hepatocellular carcinoma (394,395), presumably through systemically

disseminated metabolic networks. Helicobacter pylori is a Gram-negative bacterial pathogen that selectively colonizes the gastric epithelium. It is postulated that half of the world's

population is infected with H. pylori, though colonization of the pathogenic bacteria does

not cause any symptoms in majority of the population. Nevertheless, long-term carriage of

H. pylori significantly increases the risk of developing diseases. Among infected individuals,

approximately 10% develop peptic ulcer disease, 1% to 3% develop gastric adenocarcinoma,

and <0.1% develop mucosa-associated lymphoid tissue (MALT) lymphoma. However, at

initial stages, gastric MALT lymphoma can be cured completely by eradication of H. pylori

with antibiotics (396).

Apart from antibiotics, probiotics may also inhibit tumorigenesis and cancer progression.

Konishi et al. (397) reported that the culture supernatant of Lactobacillus casei has tumor-

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suppressive effect on colon cancer cells. The authors reported that ferrichrome produced by

L. casei is the molecule that provides tumor protection and is exerted via the JNK signaling pathway.

The etiology of breast cancer is still not understood, though it is believed the disease is due

to a combination of both genetic and environmental factors. It is posited that environmental

factors influence breast cancer as there is an increased incidence of breast cancer among

migrants and their descendants after they move from a region of low breast cancer risk to a

region of high risk. Bacterial communities within the host could be one such environmental

factor that may influence breast cancer development. Different bacterial profiles in breast

tissue exist between healthy women and those with breast cancer. Breast cancer patients had

higher levels of Bacillus, Enterobacteriaceae and Staphylococcus. E.coli and Staphylococcus epidermidis isolated from breast cancer patients induced DNA double-stranded breaks in HeLa cells. There was also a decrease in some lactic acid bacteria, known for their beneficial

health effects, including anticarcinogenic properties (398). It has been demonstrated that

women who drink fermented milk products have a reduced risk of breast cancer

development (399). Oral administration of Lactobacillus species has been shown to be protective in animal models of breast cancer (400).

Case studies back to the 1700s have recounted the development of bacterial infections in

cancer patients that led to remissions of their malignant disease. One of the pioneers in this

field, the U.S. surgeon William B. Coley, engaged in a lifelong study of this phenomenon

after the loss of his very first patient in the late 1800s to a rapidly invasive sarcoma (401).

Searching the literature available, Coley discovered records of another sarcoma patient with

relentless sarcomatous recurrences following surgical resection and an ultimate wound

infection (erysipelas) with Streptococcus pyogenes and high fever. To his surprise, after each attack of fever, the ulcer improved, the sarcoma shrank, and the lesion ultimately regressed

completely. Coley suspected that in some manner the infection had induced tumor regression

and began a series of trials to “cure” his cancer patients with pathogen inoculation. He

infected his next 10 patients, but observed intrinsic variability in efficacy using this

method(402). Due to this unpredictability, he elected to create a formulation containing two

killed bacteria: Streptococcus pyogenes and Serratia marcescens. Under the form of an inactivated vaccine, he could simulate an infection (inflammation, chills, fever) without the

actual risks of a life-threatening disease. This vaccine became known as “Coley toxins.” The

relative success with Coley's vaccine was by no means limited to sarcomas. For decades, this

vaccine form had been used by other contemporaries for carcinomas, lymphomas,

melanomas and myelomas (403,404).

However, with the advent of radiotherapy and chemotherapy, and the empowerment of the

U.S. FDA in 1964 that restricted clinical use of “Coley toxins,” the use of microbial toxins

in oncology fell out of use. There were, however, rare instances in which this line of thinking

endured and eventually received FDA approval. Perhaps the most prominent example is the

use of Bacillus Calmette-Guerin (BCG) for the treatment for superficial bladder cancer

(405). BCG is currently the only conventional bacterial vaccine in use for direct tumor

killing. Unlike Coley toxins, BCG is not administered with the ultimate goal of induced

fever. But similar to Coley's methods, the vaccine is applied directly to the tumor site with

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repeated courses following initial resection to prevent recurrence (406). After intravesicular

administration of this vaccine, a wide range of cytokines become detectable in the urine,

including interleukins-(IL)-1, IL-2, IL-6, IL-8, IL-10, IL-12, IL-18, interferon-γ, interferon- γ inducible protein-10, macrophage colony stimulating factor, and TNF-α (407-409). This inflammatory host response illustrates the point that individual immunomodulating

cytokines are partial components of a much more complex immunological response to

infection, and correspondingly, tumor regression. Some insights were gleaned from Coley's

toxins and other historical reports on live or attenuated bacterial inoculations. One was that a

local inoculation produces only a local response. Thus, BCG use is limited to superficial

bladder cancer. The heat and immune activation associated with local inflammation are

perceived to be a minimized febrile response, and correspondingly this local response is only

effective in the immediate region where it occurs (401). Since Coley's passing, the field of

tumor immunology has developed into a better founded and more sophisticated specialty,

with investigators not only employing variety of basic immunologic principles (e.g.,

antitumor cytokines, cytotoxic T cells, immune-stimulatory antibodies, cell-based vaccines)

but pivotal insights into the dominance of tumoral immune suppression in blunting the

effectiveness of any immunotherapy. Overcoming this historical source of failure in the

efficacy of cancer immunotherapy is empowering this field anew today.

There are more than a hundred chemotherapy drugs to treat many types of cancers. However,

it is not fully understood the mechanism of some of these drugs. Cyclophosphamide is a

clinically important chemotherapeutic cancer drug that stimulates anti-tumor immune

responses. Viaud et al. (410) demonstrated that cyclophosphamide alters the composition of

microbiota in the gut and induces the translocation of several Gram positive bacteria into

mesenteric lymph nodes and spleen. In the lymphoid organs the Gram positive bacteria

stimulated the generation of pathogenic T helper 17 (pTh17) cells and memory Th1 immune

responses. Germ-free tumor-bearing mice or treated with antibiotics to kill Gram positive

bacteria showed a reduction in T cell responses and their tumors were resistant to

cyclophosphamide. Adoptive transfer of pTh17 cells restored the anti-tumor efficacy of

cyclophosphamide. Overall, the study suggested that the gut microbiota help shape the

anticancer immune response.

Turning the tables: using engineered microbes to attack cancer

Over the past century, knowledge gained on how selective microbes either facilitate the

growth of cancer or alternatively act as tumoricidal agents permits us open access to utilize

this double-edged sword to our advantage. Such interventions have already begun and span

several modalities, including but not limited to the use of helper peptide sequences from

bacterial subunits, bacterial toxin-fusion proteins, oncolytic viral vaccines, and, as recent

studies elucidate, leveraging the metabolism of host flora to potentiate immune-modulators.

Broadly defined, an immunotherapeutic is any modality that manipulates the immune system

for enhanced therapeutic outcome. These include non-specific activation of the immune

system with microbial components or cytokines; antigen-specific adoptive immunotherapy

with antibodies or lymphocyte transfers; and active immunotherapy by direct vaccination

against tumor-specific proteins, or antigens. We traditionally regard vaccines as educators

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for the naïve immune system that are administered prophylactically in anticipation of any

infection. However, in the setting of aggressive cancers, the use of cellular vaccines to mount

reactive immune responses is now being employed with promising results.

A variety of cancer vaccines are currently under investigation, but perhaps the most widely

investigated to date are 1) cellular vaccines composed of APC loaded with tumor antigen

(411-414) and 2) peptide vaccines (415,416). Peptide vaccines are comprised of 8–25 amino

acids that encompass an epitope - a recognizable sequence coding for an antigen. The

transient nature and low magnitude of responses in many cancer patients has elucidated that

tumors themselves are inherently proficient at downplaying immune responses as well as

escaping antigen recognition altogether. Thus, there is an urgent need for improving vaccine

immunogenicity and for ensuring that cancer antigens are sufficiently immunogenic. To

enhance peptide vaccine immunogenicity, these small peptides are often conjugated to a

carrier protein, such as keyhole limpet hemocyanin (417,418) and tetanus toxoid (419,420).

These helper proteins enable recognition by and activation of the immune system with great

potency and generate complimentary bystander activation with cytokine release and

maturation of effector cell phenotype. Peptide vaccines are appealing in cancer therapy

because they are relatively easy to manufacture and store, and they do not require laborious

preparations. Due to their “off-the-shelf” feature, repeated boosting for enhanced immune

activation also distinguishes peptide cancer vaccines as an expandable modality (421).

Adoptive immunotherapy has also been utilized to eradicate established tumors (422). This

process involves ex vivo activation of autologous immune cells, isolated from either

peripheral blood or intratumoral lymphocytes (423,424), into lymphocyte-activated killer

cells. Lymphocyte-activated killer cells are generated by culturing autologous peripheral

lymphocytes with IL-2, a vital growth cytokine for generating T cells and NK cells. These

killer cells are then returned to the patient intratumorally or intravenously, where they

become activated by host APCs and exert their tumoricidal effects. Using the knowledge we

have gained from microbial components, NK cells have been potentiated by leveraging the

cytolytic capacity of microbial diphtheria toxin. One study demonstrated that haploidentical

NK cells for relapsed and refractory acute myeloid leukemia could be augmented and

improved with a lymphodepletive platform using diphtheria toxin conjugated to IL-2. Using

the immunotoxin IL2DT to deplete immunosuppressive Treg, investigators appreciably

improved rates of in vivo NK-cell expansion (10% versus 27%) and AML 28-day remission (53% vs 21%; P= 0.02) compared to the cohort without IL2DT (425).

Oncolytic viruses represent another immunotherapy modality that has gained recent traction

in the field of tumor immunology. Use of these microbes was first based on early reports of

spontaneous cancer remissions coincident with natural infection or upon the use of live

attenuated vaccines (426). Since then, an improved understanding of the molecular basis for

viral host cell tropism, cytotoxicity, and cell type-specificity has opened up avenues for the

very selective design of virally-based anti-cancer strategies. To be efficacious and safe, an

oncolytic virus must possess 1) an inherently low human pathogenic potential (i.e. the

orphan reovirus [(427)]), 2) a veterinary pathogen with unknown human pathogenicity (i.e.

vesicular stomatitis virus [(428)]), or 3) a human pathogen genetically engineered to

selectively kill cancerous cells without collateral cytotoxicity in normal cells (i.e. herpes

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simplex virus-1 [(429)]). One such group is utilizing a prototype nonpathogenic poliovirus

recombinant, known as PVSRIPO. Poliovirus naturally targets the vast majority of

ectodermal/neuroectodermal cancers expressing its cellular receptor, CD155. Evidence from

glioblastoma patients suggests that the CD155 receptor is ectopically upregulated on tumor

cells. Preclinical studies have shown that treatment of glioma xenografts with intratumoral

inoculation of PVS-RIPO produced rampant tumor cell death, potent host-mediated

inflammatory reactions against infected tumors, and rapid tumor decline (430,431). The use

of PVS-RIPO is now being evaluated in Phase I setting for recurrent glioblastoma (432).

As multiple studies are being carried out yearly, investigators continue to uncover key

barriers to efficacy that further clarify our strategies. As with any antigen-specific immune

response, several homeostatic mechanisms remain at play to prevent rampant damage to the

host or autoimmune toxicity. One of the most exciting regulatory axes to be studied recently

is that of programmed death (PD)-1/PD-ligand-1 (PD-L1). PD-1 is a co-inhibitory receptor

that is inducibly expressed by T and B cells upon activation. Antigen-specific T cells

expressing the PD-1 receptor will engage with either of its ligands, PD-L1 or PD-L2

expressed on APC, which elicits an inhibitory cascade and subsequent inhibition of T cell

receptor (TCR)-induced cytokine production and proliferation (433). PDL-1 is expressed in

several other cell types, including tumor cells and some epithelial cells, lymphoid cells, and

myeloid cells (434). Another axis involved in regulating self-recognition is that of cytotoxic

T-lymphocyte antigen-4 (CTLA-4). Studies have demonstrated that tumor cells stimulate

CTLA4, promoting a cascade of inhibitory immune processes and ultimate T cell inactivity

against tumors themselves (435,436).

In cancer immunotherapy, monoclonal antibodies (mAb) against the immune checkpoints

CTLA-4, PD-1 and its ligand PD-L1 have demonstrated high activity in melanoma and other

tumors (437). Ipilimumab, an anti-CTLA-4 antibody, was the first approved “immune

checkpoint inhibitor.” Although the response rate with ipilimumab is low (less than 20% of

patients have objective responses), many of those positive responses were associated with

long-term survival (438), with similar results in the first and second line settings. Nivolumab

and pembrolizumab, both anti-PD-1 inhibitors, have now also been approved for the

treatment of melanoma, with response rates of up to 40% and a demonstrated survival

advantage in Phase III trials (434).

Strikingly, recent findings in preclinical models of cancer stress the importance of intact gut

microbiota for effective immune checkpoint blockade. One recent study found that

antitumor effects of CTLA-4 blockade depended on the presence of distinct Bacteroides species. In both mice and patients, T-cell responses specific for B. thetaiotaomicron or B. fragilis were associated with the efficacy of CTLA-4 blockade. Using antibiotic-treated as well as germ-free mice, tumors lacking these strains did not respond to CTLA blockade.

This deficiency was rescued by B. fragilis gavage, by immunization with B. fragilis polysaccharides, or by adoptive transfer of B. fragilis–specific T cells. Ultimately, fecal microbial transplantation from humans to mice confirmed that treatment of melanoma

patients with antibodies against CTLA-4 favored the outgrowth of B. fragilis with anticancer properties (439). A different preclinical study similarly elucidated that mice treated with gut

commensals of Bifidobacterium displayed significantly improved suppression of melanoma

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growth in comparison with non-Bifidobacterium treated counterparts. These observed differences in spontaneous antitumor immunity were eliminated upon co-housing or after

fecal transfer, eluding to the importance of shared bacterial colonization. Furthermore,

administration of the bacteria to Bifidobacterium-naïve mice with established melanoma significantly enhanced tumor-specific immunity and response to anti-PD-L1 mAb therapy

(440). Although the mechanisms from both these studies are not fully understood, they laid

the foundation behind the requirement for an intact microbiome to enact antitumor

responses. One key observation from both these studies was that they employed

subcutaneous tumor models, meaning that intestinal microbiota exerted antitumor immunity

in a systemic fashion. Both studies relied on the presence of CD8+ T cells. Secondly, both

demonstrate that altered DC activation was a responsible intermediate event between the

presence of gut microbiota and provision of checkpoint inhibitors (Fig. 4).

Conclusions

It is humbling to consider how much biomedical research has been conducted since the

molecular biology revolution of the past century without appreciation of the importance of

microbiomes in health and disease. Like all realms of biomedical investigation, the field of

cancer research can no longer ignore the ‘other half’ of the organism; it must become as

familiar with the genetics, biology, physiology and immunologic effects of host

microorganisms as with the hosts themselves. In considering sources of experimental

irreproducibility in biomedical publications that have been suggested recently to be

disturbingly high, it seems likely that natural variations in microbiome infections present in

experimental models and vivariums at different sites will provide one more challenge to the

exquisitely difficult problem of how one defines a “molecular mechanism” in disease. More

focus on practical applications (sought by most funding organizations) along with empirical

explorations once traditional to biology may offer two paths forward, since, to paraphrase

the pragmatic American philosopher Charles S. Peirce, “You know something if you can do

something.” How the present obsession with molecular mechanism will change in the face

of the challenge the microbiome poses to preclinical research is unclear. Nevertheless, as the

molecular biology revolution continues to wash up on the shores of reductionism this

century, it will be impossible not to re-embrace the roots of traditional biological thought,

where ecology, evolution and a focus on emergent principles in complex organisms can help

re-center the pursuit of new knowledge and its applications to improve disease management

and healthful lifespans.

Acknowledgments

The laboratories of Jacques Izard are supported by National Institutes of Health (NIH) grants R01CA202704 and Nebraska Tobacco Settlement Biomedical Research Development Funds. The APC Microbiome Institute is supported by Science Foundation Ireland (SFI) (Grant Number SFI/12/RC/2273) and has conducted studies in collaboration with several companies including GSK, Pfizer, Wyeth and Mead Johnson. Dr Clarke's contribution to this review was specifically supported by the Irish Health Service Executive (Grant number HaPAI/2015/GC). Sunil Thomas, Alexander J. Muller, James M. Mullin, George C. Prendergast acknowledge support from the Lankenau Medical Center Foundation, the Women's Board of Lankenau Medical Center and Main Line Health. The content of this review was neither influenced nor constrained by funding from these Organizations.

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Figure 1. Early endosomes localize mainly to the periphery of colon mucosa and muscularis (A) Peripheral localization in a colony of human colonic Caco-2 cells. Cells were stained

with the early endosomal marker EEA1 (green) or DAPI to visualize cell nuclei (blue) and

processed for immunofluorescence microscopy. (B) Peripheral localization in murine colon

mucosa processed as above. (C). Peripheral localization in murine colon muscular processed

as above.

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Figure 2. Late endosomes locate throughout colon mucosa and muscularis (A) Punctate cytosolic localization in a colony of human colonic Caco-2 cells. Cells were

stained with Beclin1, an autophagic regulator associated with late endosomes (green), or

DAPI to visualize cell nuclei (blue) and processed for immunofluorescence microscopy. (B)

Cytosolic localization in murine colon mucosa processed as above. (C). Cytosolic

localization in murine colon muscularis processed as above.

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Figure 3. Dysbiosis: an immunocompromised state characterized by pathobiont colonization that leads to hyperinflammation, dysplasia and tumorigenesis (Left) Symbiosis: A symbiotic gut microbiota operates under a functional intestinal

epithelial cell (IEC) barrier, with steady state proportions of mucus, pattern recognition

receptors (PRRs), antimicrobial peptides, and secretory IgA, which in turn contain the

microbiota in the intestinal lumen. Under tight control by IECs, the intestinal immune

system within the gut lamina propria becomes largely tolerant to the resident commensals.

Signaling cascades that occur downstream of toll-like receptors (TLRs) are used by IECs to

detect microbes through PRRs. Upon lipopolysaccharide (LPS) stimulation of TLRs, the

MYD88 protein is recruited, activating the NF-κB pathway, leading to production of antimicrobial proteins and proinflammatory cytokines. In a symbiotic gut, IECs are

desensitized by repeated exposure to LPS or are attenuated by LPS-mediated

downregulation of the IL-1 receptor–associated kinase 1 (IRAK1), an activator of the NF-κB cascade. Exposure to LPS induces epithelial cells to secrete TGF-β, B-cell-activating factor of the TNF family (BAFF), and a proliferation-inducing ligand (APRIL), all of which

promote the development of tolerogenic responses to the microbiota. CD103+ dendritic cells

(DCs) support the development of regulatory T (Treg) cells to secrete IL-10 and TGF-β, and together they stimulate the production of commensal-specific IgA. (Right) Dysbiosis:

Increased intestinal exposure of diverse PAMPs, pro-inflammatory cytokines, apoptotic

debris, and toxins leads to microbial dysbiosis and overgrowth of “pathobionts”, transformed

symbiotic bacteria now under pathologic conditions. Pathobiont overgrowth leads to the loss

of barrier integrity and a breach in the IEC barrier. Translocation of bacteria and bacterial

components triggers the intestinal immune system through TLR activation, resulting in

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potentially harmful effector T cell responses set to clear invading bacteria. Ultimately, the

secretion of IL-1 and IL-6 from IECs fuels a TH1 and TH17 response by DCs and

macrophages and leads to higher levels of commensal-specific IgG by B cells.

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Figure 4. Gut microbiome directs the efficacy of immune checkpoint therapy Both anti-CTLA-4 and anti-PD-L1 therapies rely on gut microbiota for efficacy in immune

activation. Anti-PD-L1 therapy has been shown to rely on the pre-existence of sufficient

Bifidobacterium species, which are also thought to augment responses via PD-L1 binding on antigen-presenting cells such as DCs and macrophages. Subsequent ligation results in the

prevention of suppressive signals to PD-1-expressing T cells. Similarly, anti-CTLA-4

indirectly alters the intestinal flora and enriches the Bacteroides species, possibly by promoting deterioration of the IEC barrier via activation of local lymphocytes. These

bacteria then promote the activation of DCs, which present tumor antigens to prime and

maintain anti-tumor T cell responses. Anti-CTLA-4 holds additional activation functions,

including 1) preventing CTLA-4 from blocking activation of the co-stimulatory molecule

CD28 on T cells and 2) blocking the immune-suppressive function of Tregs, which are

required in deactivation of immune responses against tumors.

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