Dendritic Cells

Intestinal dendritic cells are located within specific intestinal lymphoid tissues, collectively termed GALTs, or diffusely distributed throughout the intestinal lamina propria. Dendritic cells are very important cells that act as sensors of microbial ligands through activation of innate immune receptors (eg, toll-like receptors [TLRs] and c-type lectin receptors). For example, a Lactobacillus rhamnosus bacterial strain is recognized by dendritic cell TLR-2 and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin. The signaling pathways triggered by bacterial-derived molecules allow for changes in dendritic cell phenotypes and cytokine secretion, which polarize the subsequent adaptive T-cell immune response into T helper 1 (T _H 1), T _H 2, T _H 9, T _H 17, T _H 22, or T regulatory cells (T regs). Thus, appropriate dendritic cell recognition of microbial factors is key to the integration of microbial and/or host metabolism with immune functions ( Fig. 1 ).

Dendritic cell activation by microbes polarizes the adaptive immune response. Dendritic cells recognize microbial components and metabolites via PRRs (eg, toll-like receptors [TLRs], Nucleotide-binding oligomerization domain [NOD]) and GPCRs (eg, histamine receptor 2). Following activation, dendritic cells present antigen, alter cell surface expression of costimulatory or inhibitory molecules, and release mediators, such as cytokines or metabolites, which shape the subsequent adaptive immune response. DC, dendritic cell; GPCR, G protein-coupled receptor; IDO, Indoleamine-pyrrole 2,3-dioxygenase; IL, interleukin; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PD-L1, Programmed death-ligand 1; RA, retinoic acid; TNF-α, tumor necrosis factor alpha.

Metabolism of vitamin A to retinoic acid is a key immunomodulatory activity associated with intestinal dendritic cells. Certain, but not all, intestinal microbes can induce retinoic acid metabolism by human dendritic cells in vitro and by murine CD103 ⁺ dendritic cells within the small intestine lamina propria. In contrast, Helicobacter pylori infection severely disrupted gastric retinoic acid biosynthesis, which could impair dendritic cell retinoic acid signaling and may contribute to disease progression. In addition to vitamin A metabolism, induction of another dendritic cell metabolic enzyme, heme oxygenase-1, was shown to be required for induction of mucosal T regs within mesenteric lymph nodes by Lactobacillus rhamnosus .

The influence of the microbiota on innate immune cells has been shown to affect the host response to cancer therapy. For example, germ-free mice and mice that are treated with antibiotics both show a diminished response to immunotherapy by CpG oligonucleotides and chemotherapy owing to the impaired function of myeloid-derived cells in the tumor microenvironment. Furthermore, bifidobacteria enhance immunity to tumors through the augmentation of dendritic-cell function leading to better control of implanted syngeneic tumors by CD8-expressing T cells. These studies open up a fascinating avenue of research whereby specific members of the microbiome may promote antitumor innate immune responses that are required for cancer therapies to be successful.

Macrophages

Macrophages are professional myeloid phagocytes and are highly specialized in removal of host cellular debris, foreign substances, microbes, and cancer cells. Myeloid cells modulate important pathways, such as interleukin 22 (IL-22) production by innate lymphoid cells (ILCs), which induces the production of epithelial regenerating islet-derived protein 3 (RegIII)β and RegIIIγ, antimicrobial peptides that are important for maintaining a spatial separation between most commensal bacteria and the intestinal epithelial layer; this modulation is also pivotal for the local containment of commensals. Local concentrations of microbiota-derived metabolites, as well as systemic levels of microbial products, seem to drive myeloid-cell differentiation and function through PRR signaling. Indeed, these microbiota-driven alterations in the myeloid-cell pool greatly influence the susceptibility of the host to a variety of disorders, which range from infection to allergy and asthma. Intestinal microbial colonization drives the continuous replenishment of macrophages in the intestinal mucosa by monocytes that express C-C chemokine receptor type 2. Microbial sensing by myeloid cells within the lamina propria provides regulatory signals that are crucial for the maintenance of commensal mutualism and the initiation of inflammatory responses in the host.

Innate Lymphoid Cells

ILCs, a recently discovered lymphocyte branch of the innate immune system, develop normally in the absence of the microbiota; but the proper functioning of ILCs depend on commensal microbial colonization. Similarly to T lymphocytes, which are classified according to their cytokine secretion and transcription factor expression, ILCs have been classified as ILC1, ILC2, and ILC3. Most studies that examine the influence of the microbiota on ILCs have focused on ILC3. The importance of ILC3 cells in host-microbiota interactions became clear when their depletion and the resulting abrogation of IL-22 production was shown to produce a loss of bacterial containment in the intestine. However, other studies have reported elevated secretion of IL-22 by ILCs in the absence of the microbiota, whereas further studies have documented the abrogation of IL-22 secretion using antibiotics. One part of this response, which may be important, is flagellin sensing by CD103 ⁺ myeloid cells that drives IL-23-mediated production of IL-22 by ILCs. In addition, the presentation of microbial antigens by ILC3s limits commensal-specific T-cell responses, which maintain tolerance to commensal bacteria. An equally important microbiota-instructed function of ILCs is their communication with epithelial cells. Microbiota-induced IL-22 production by ILC3s induces expression of the enzyme fucosyltransferase 2 (galactoside 2-α- l -fucosyltransferase 2) and fucosylation of surface proteins by intestinal epithelial cells, which is required for host defense against enteric pathogens. Indeed, this pathway may function to promote the growth of beneficial members of the microbiota and contribute to the maintenance of a stable community of microorganisms, as during the starvation associated with intestinal infection, the shedding of fucosylated proteins into the intestinal lumen serves as a source of energy for commensal bacteria. The microbiota might also influence the activity of the other ILC subsets, such as ILC2s, which are activated by epithelial cell–derived IL-25 produced in a microbiota-dependent manner.

Epithelial Cells

Although not classically considered to be bona fide members of the innate immune system, intestinal epithelial cells are equipped with an extensive repertoire of innate immune receptors. However, the most important job of the epithelial cell is nutrient absorption. Pathogen infection can reduce epithelial cell digestive enzyme activity; but this detrimental effect can be reversed by certain commensal microbes, possibly via modulation of mucosal inflammatory responses. In addition to their absorptive function, epithelial cells form a mucosal barrier that protects host tissue from damaging agents such as luminal pathogens and toxic products. One protective barrier mechanism is the production and secretion of antimicrobial peptides, such as defensins and cathelicidins. Gut microbes have been shown to differentially regulate defensin expression and protein secretion, which is influenced by local inflammatory mediators. Metabolites from the microbiome induce epithelial cell inflammasome signaling resulting in IL-18 secretion and antimicrobial peptide production. Interestingly, host genetic deficiency in this pathway results in dysbiosis; this dysbiotic microbiome can hijack the microbiome of wild-type animals. Autophagy is an important adaptive response to stress, which promotes cell survival and is required for maintenance of the epithelial barrier. Several bifidobacteria have been recently described that promote autophagy in an intestinal cell line. The mucous layer coating the gastrointestinal tract is an important barrier component, and gut bacteria have been shown to promote mucin production by goblet cells in the intestine. Recently, a protein called p40 from a lactobacillus strain was demonstrated to be sufficient for stimulation of mucin production through transactivation of the epithelial epidermal growth factor receptor.

Excessive epithelial cell responses to microbial ligands result in local inflammatory responses, which disrupt the epithelial barrier. The presence of certain microbes can actually protect the epithelial cells and dampen the epithelial cell cytokine and chemokine responses. However, not all chemokine responses are impacted to the same extent by every bacterial strain; a single bacterial strain may reduce expression of certain chemokines, while increasing the expression of others. For example, Bifidobacterium bifidum PRL2010 suppresses CCL22 expression but enhances CCL19 expression, suggesting that strain-specific and chemokine-specific responses are induced by gut bacteria.

The PRR nucleotide-binding oligomerization domain-containing protein 2 (NOD2), which is highly expressed in the Paneth cells of the small intestine, is activated by microbial peptidoglycans present within the gut and generates a cellular response that includes the secretion of cytokines, the induction of autophagy, intracellular vesicle trafficking, epithelial regeneration, and the production of antimicrobial peptides, thereby influencing epithelial health and the composition of the microbiota. Epithelial cell NOD1 expression is important for both the C-C motif chemokine 20–mediated generation of isolated lymphoid follicles in the intestine and control of bacterial colonization. NOD1 is one of the important sensors that regulates the communication between the host and microorganisms through the production of inflammasome-mediated IL-18 and the downstream expression of antimicrobial peptides, and it also controls the secretion of mucus by goblet cells.

T Lymphocytes

The beneficial effects of microbiome interactions with the host immune response against diseases, such as allergy or colitis, are often associated with enhancement of T reg cells. The main mechanisms underpinning T reg cell effects include production of inhibitory cytokines (IL-10, transforming growth factor [TGF]-β. and IL-35), effector cells cytolysis (via secretion of granzymes A and B), direct targeting of dendritic cells via inhibitory programmed cell death protein 1 and cytotoxic T-lymphocyte-associated protein 4 cell surface molecules, and metabolic disruption of effector cells (CD25, cAMP, adenosine, CD39, and CD73) and T reg cells that are induced by bacteria can have systemic antiinflammatory functions. The relative contribution of individual members or defined communities within the gut microbiota in the accumulation and functional maturation of T reg cells in the intestine is starting to be documented. Clostridia strains that fall within clusters IV, XIVa, and XVIII seem to have a strong capacity for inducing the accumulation of T reg cells in the colon. Clostridia from this cluster also stimulate ILC3s to produce IL-22, which helps to reinforce the epithelial barrier and reduces the permeability of the intestine to dietary proteins. Mice colonized by a microbiota that includes Clostridia display a suppressed response to food allergens. Many other commensal microbes, such as Bifidobacterium longum , Lactobacillus reuteri , and Lactobacillus murinus , have been also shown to increase the proportion of T reg cells in mice. Notably, consumption of B longum 35624 by healthy human volunteers resulted in an increased proportion of FOXP3 T reg cells in peripheral blood, whereas administration of this probiotic to patients with psoriasis, chronic fatigue syndrome, or ulcerative colitis consistently resulted in reduced levels of serum proinflammatory biomarkers, such as C- reactive protein, possibly mediated by increased numbers of T reg cells.

Crosstalk between innate and adaptive cells to microbial signals promotes immune homeostasis in the intestine, including the induction of T regs. A recent study has revealed how complicated this crosstalk can be. Microbial signals sensed by intestinal macrophages resulted in IL-1β secretion, and this IL-1β promoted granulocyte macrophage colony-stimulating factor (GM-CSF) release by local ILC3 cells. ILC3-derived GM-CSF then triggers dendritic cell and macrophage secretion of retinoic acid and IL-10, which, in turn, promotes the induction and expansion of T regs. Disturbance of this crosstalk significantly altered mucosal immune effector functions, resulting in impaired oral tolerance to dietary antigens. Interestingly, more severe allergic responses to food allergen challenge was observed in gnotobiotic mice reconstituted with microbes from allergic animals. In this animal model, allergic responses were associated with a decreased abundance of Firmicutes and increased abundance of Proteobacteria. In contrast, Proteobacteria seemed to be less abundant in infants with atopic symptoms, which was hypothesized to be related to the lipopolysaccharide (LPS) incorporated into the bacteria cell wall. LPS drives T _H 1 polarization by enhancing IL-12 secretion, which may dampen T _H 2-dominant responses in atopic individuals. Collectively, there is considerable overlap between the responses of T reg cells to many (but not all) microbes present within the gut, which indicates that different cellular and molecular pathways supporting T reg generation and survival converge in the intestine. The induction and maintenance of T reg cells might be a common and crucial mechanism for maintaining the homeostatic and beneficial relationship between the microbiota and the host.

T _H 17 cells secrete IL-17 that protects mucosal cells from infection, but exaggerated activity of this cell subset induces tissue inflammation. It was recently shown that the gut microbiota induces dendritic cells and macrophages to produce IL-1β and IL-6, which both drive T _H 17 differentiation and arthritis. The number of T _H 17 cells in the intestines varies widely between animal facilities, even in genetically identical mice that have been reared in specific pathogen-free conditions, and often reflects whether mice have been colonized with segmented filamentous bacteria (SFB), and SFB also promote fucosylation of the epithelium through the activation of ILC3 cells. On the other hand, gut-derived commensal bacteria have also been shown to suppress T _H 17 responses both through direct and indirect mechanisms. T _H 17/IL-17 activity can be suppressed by inducing T reg and/or T _H 1 cell subsets, by induction of IL-27 production, which suppress the generation of IL-17 and induce IL-10 or by stimulation of TLR-9 on T _H 17 cells. These data suggest that the commensal microbiota is important for inducing both proinflammatory and regulatory responses in order to rapidly clear infections and minimize inflammation-associated tissue damage.

Natural Killer T Cells

Natural killer T (NKT) cells are central mediators of intestinal inflammation; pathogenic NKT cell activation is mediated by CD1d ⁺ bone-marrow-derived cells, whereas CD1d ⁺ epithelial cells protect against intestinal inflammation. It has been shown that NKT cells can be influenced by the gut microbiome, and microbial lipid antigens may directly activate NKT cells. Recently a healthy human volunteer study showed that the combination of xylo-oligosaccharide with Bifidobacterium animalis reduced CD16/56 expression on NKT cells. However, the functional consequences of altered NKT cell activation by the microbiome in humans remain to be determined.

B Cells

B lymphocytes have an essential role in humoral immune responses via their secretion of antigen-specific antibodies. In addition, B cells can limit aggressive immune reactivity. B cells regulate immune responses mainly via IL-10, which has been shown in experimental models of infection, allergic inflammation, and tolerance. Indeed, the gut microbiota is important in driving the differentiation of IL-10–producing B regulatory cells.

B cell–dependent modulation of the microbiome was shown in immunoglobulin A (IgA)–deficient mice. IgA-deficient mice had persistent intestinal colonization with γ-Proteobacteria that cause sustained intestinal inflammation and increased susceptibility to neonatal and adult models of intestinal injury. The group also identified an IgA-dependent mechanism responsible for the maturation of the intestinal microbiota in mice. Recently another group showed that the number of gut-homing IgG+ and IgA+ B cells was significantly higher in infants compared with adults. This finding suggests that activation of naïve B cells in the gut overlaps with the establishment of the gut microbiota in humans. Oral administration of Lactobacillus gasseri SBT2055 (LG2055) induced IgA production and increased the number of IgA+ cells in Peyer patches and in the lamina propria. Combined stimulation of B cells with B-cell activating factor and LG2055 enhanced the induction of IgA production. Clostridia-induced T reg cells support the production of IgA in the intestine, which contributes to increased diversity of the microbiota and, in particular, of Clostridia. Communication between B cells and other cell types is important for IgA production, as the secretion of lymphotoxin-α (also known as tumor necrosis factor-β) by ILC3s is crucial for the production of B-cell IgA and for microbiota homeostasis in the intestine. In the absence of IgA, the commensal bacterium Bacteroides thetaiotaomicron , which typically does not trigger inflammation in the human gut, expresses high levels of gene products that are involved in the metabolism of nitric oxide and elicits proinflammatory signals in the host. IgA that has undergone affinity maturation through somatic hypermutation binds to and selects for particular components of the microbiota, which leads to an increase in the diversity of the microbial community and enhances mutualism between the microbiota and the host. Consistent with this observation, people who are deficient in IgA have more bacteria from taxa with potentially inflammatory properties. Mice that are deficient in T cells owing to a lack of T-cell antigen receptor chains β and δ, as well as those that lack T follicular cells and the T-cell-dependent IgA pathway owing to T-cell-specific inactivation of the gene Bcl6 in CD4+ T cells, retain an IgA-mediated response that is specific to antigens from commensal bacteria, indicating that T-cell–independent B-cell IgA production is directed at the microbiota. However, this response is characterized largely by the low-affinity binding of IgA to antigens that are shared by multiple species of bacteria. Thus, B-cell–derived IgA plays an important role in host defense against mucosally transmitted pathogens, controls the adherence of commensal bacteria to epithelial cells, and neutralizes bacterial toxins to maintain homeostasis at the mucosal surfaces.