Functions of innate immune cells and commensal bacteria in gut homeostasis

Abstract

The intestinal immune system remains unresponsive to beneficial microbes and dietary antigens while activating pro-inflammatory responses against pathogens for host defence. In intestinal mucosa, abnormal activation of innate immunity, which directs adaptive immune responses, causes the onset and/or progression of inflammatory bowel diseases. Thus, innate immunity is finely regulated in the gut. Multiple innate immune cell subsets have been identified in both murine and human intestinal lamina propria. Some innate immune cells play a key role in the maintenance of gut homeostasis by preventing inappropriate adaptive immune responses while others are associated with the pathogenesis of intestinal inflammation through development of Th1 and Th17 cells. In addition, intestinal microbiota and their metabolites contribute to the regulation of innate/adaptive immune responses. Accordingly, perturbation of microbiota composition can trigger intestinal inflammation by driving inappropriate immune responses.

In the intestine, a refined balance is maintained between inflammatory responses and tolerance against multiple environmental factors, including microflora and food antigens. This is because aberrant inflammatory responses can lead to the development of inflammatory bowel disease (IBD), such as Crohn’s disease (CD) and ulcerative colitis. The number of effector T cells such as T helper (Th)1 cells and Th17 cells is increased in the gut mucosa of patients with IBD compared with healthy individuals, suggesting that Th1/Th17 responses contribute to the development or/and pathogenesis of IBD (1). Thus, the number and activity of effector T cells are tightly regulated by several mechanisms in the gut mucosa. For example, Foxp3⁺ regulatory (T_reg) cells, abundantly present in the intestinal lamina propria, suppress inflammatory responses by producing anti-inflammatory cytokines including interleukin (IL)-10 and transforming growth factor (TGF)-β. IL-10 derived from T_reg cells is thought to regulate the activity of intestinal myeloid cells.

Recently, several innate immune subsets possessing the ability to modulate intestinal homeostasis were identified in human and murine intestinal mucosa (2–4). In the gut, the activity of innate immune cells such as dendritic cells (DCs) and macrophages is tightly regulated by several mechanisms, and the excessive and inappropriate initiation of innate immunity causes development of IBD. Some innate myeloid subsets have been reported to promote intestinal inflammation by colitogenic effector T cells through upregulation of gut homing receptor α4β7 (5) and production of colitogenic cytokines such as IL-6 and IL-23 via high expression of TLRs (6). Accordingly, patients with CD have abnormal innate immune cell functions, including cytokine production, pathogen clearance and recruitment of neutrophils. This indicates that intestinal innate immunity is responsible for maintaining gut homeostasis and dysregulation of innate cell activity can lead to the development of IBD by driving colitogenic effector T cells.

Accumulating evidence has revealed that microbiota play a crucial role in maintaining gut homeostasis by controlling nutritional metabolism, epithelial barrier integrity and host immunity. Alterations of the microbial community structure, termed dysbiosis, are implicated in IBD because of increased pathogen invasion and the overgrowth of pathobionts (7–9).

In this review, we focus on a variety of intestinal innate immune subsets, which are implicated in the maintenance of gut homeostasis, and the impact of microbiota on development of host immune systems.

Innate Immunity and Gut Homeostasis

Several studies have identified a variety of mononuclear phagocytes that maintain gut homeostasis by enhancing or suppressing T cell responses (10–13). In particular, CD103⁺ DCs and CX₃CR1⁺ cells have been well characterized in the murine intestinal mucosa (14–16) (Fig. 1). In addition, the human counterparts to murine intestinal macrophages, CD103⁺ DCs and CX₃CR1⁺ cells were recently identified (Fig. 2).

Fig. 1.

Murine innate immune subsets in the gut mucosa. (A) CD103⁺ DCs facilitate the differentiation of Foxp3⁺ T_reg cells through the production of retinoic acid and TGF-β. (B) TLR5⁺CD103⁺CD11c⁺ DCs can induce the development of Th1/Th17 cells and IL-22 production in innate lymphoid cells by secreting IL-23 followed by antimicrobial peptide expression in intestinal epithelial cells. (C) CX₃CR1^intermediateCD70⁺CD11b⁺ DCs promote Th17 cell development by producing IL-6 and TGF-β upon ATP stimulus produced by commensal bacteria. (D) CX₃CR1^highM_reg cells can prevent intestinal inflammation by inhibiting effector T cell proliferation. (E) Macrophages suppress production of pro-inflammatory cytokines by myeloid cells due to IL-10 production.

Fig. 2.

Human innate immune subsets in the gut mucosa. (A) CD103⁺SIRPα^high DCs can induce Foxp3⁺ T_reg cells. (B) CD103⁺SIRPα^high DCs and CD103⁺SIRPα^- DCs initiate Th 17 cell development. (C) CD14⁺CD163^low cells induce Th17 differentiation by expression of IL-6, IL-23p19, TNF-α and IL-1β via TLR2, TLR4, and TLR5. (D and E) Colitogenic IL-17 and IFN-γ-producing T cells are induced by CD14⁺CD163^low cells and macrophages in the gut mucosa of patients with IBD.

(i) CD103⁺ DCs

In the murine intestine, CD103⁺ DCs possess a variety of functions, including: inducting cytotoxic T lymphocytes (17); CD4⁺ and CD8⁺ T cell proliferation (17, 18) and gut immune tolerance by facilitating differentiation of Foxp3⁺ T_reg cells through the production of retinoic acid and TGF-β (12, 19, 20). In addition, Toll-like receptor (TLR) 5-activated CD103⁺CD11c⁺ DCs induce the development of Th1/Th17 cells (21). Adequate Th1 and Th17 responses mediate host defence against invaded pathogens. Th1 cells-produced interferon (IFN)-γ activates macrophages to kill intracellular pathogens (22) whereas Th17-related cytokines such as IL-17 and IL-22 promote the production of anti-microbial peptides (23–25) and regulate integrity of epithelial cells (26, 27), respectively. Moreover, flagellin-dependent IL-23 production by CD103⁺ DCs induces IL-22 production from innate lymphoid cells followed by antimicrobial peptide expression in intestinal epithelial cells (28). Furthermore, common probiotic, Bifidobacterium breve activates intestinal CD103⁺ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine (29).

Human CD103⁺ DCs as well as murine CD103⁺ DCs induce gut homing receptors such as CCR9 and integrin α4β7 on T cells (18). In addition, transcription factor IRF4-expressing CD103⁺CD141^-SIRPα^high DCs induce Foxp3⁺ T_reg cells by producing retinoic acid in human and murine small intestines (30). Both CD103⁺CD141⁻SIRPα^high DCs and CD103⁺CD141⁻SIRPα^- DCs initiate the development of Th 17 cells, which are equivalent to murine CD103⁺CD11b⁺ DCs that promote effector T cell differentiation.

Together, CD103⁺ DCs contribute to the maintenance of gut homeostasis by inducing immune tolerance to intestinal antigens, while promoting protective immune responses through the induction of Th1/Th17 cells in human and murine intestine.

(ii) Th17-inducing myeloid cells

Currently, several subsets of CX₃CR1⁺ cells have been characterized in the murine lamina propria, including CD11c^-CX₃CR1⁺, CD11c⁺CX₃CR1⁺CD68⁺F4/80⁺, and CD11c⁺CX₃CR1⁺CD68⁻F4/80^- cells (16). CX₃CR1⁺ cells contribute to the induction of oral tolerance by transferring fed antigens to CD103⁺ DCs via gap junction molecule connexin 43 (31). In addition, CX₃CR1⁺ cells help Th17 cells development (32, 33). In particular, CX₃CR1^intermediateCD70⁺CD11b⁺ DCs drive Th17 cell development by producing IL-6 and TGF-β upon ATP stimulus, which is derived from commensal bacteria (32). In the human intestinal mucosa, HLA-DR^highCD14⁺CD163^low cells were identified as human counterparts to murine CX₃CR1^intermediateCD70⁺CD11b⁺ DCs. These cells induce Th17 cell differentiation by producing high levels of IL-6, IL-23p19, tumour necrosis factor (TNF)-α, and IL-1β via TLR2, TLR4 and TLR5 signalling pathways. Furthermore, the Th17 cell-inducing activity of HLA-DR^highCD14⁺CD163^low cells is increased in patients with CD, suggesting that this cell subset might play a crucial role in the pathogenesis of CD.

In the intestine, commensal bacteria including Enterococcus mundtii, Enterococcus gallinarum, Escherichia coli and Salmonella secrete ATP, thereby mediating several immune responses (34–36). Thus, extracellular ATP is tightly regulated by ATP-hydrolyzing ecto-enzymes on epithelial cells and immune cells, such as ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) in the intestine. For example, E-NTPD7 on epithelial cells contributes to inhibiting Th17 development through ATP hydrolysis in the small intestine (37). In addition, E-NPP3 on mast cells is responsible for preventing allergen-induced diarrhoea by hydrolyzing ATP. ATP is secreted by mast cells upon FcεRI stimulation; thereby IL-6 production is elevated through purinergic receptor P2X7. In this context, E-NPP3 is also induced in mast cells and contributes to inhibiting ATP-dependent inflammatory responses (38).

(iii) CX₃CR1^high regulatory myeloid cells

In the murine colonic lamina propria, CX₃CR1^highCD11b⁺CD11c⁺ cells termed regulatory myeloid (M_reg) cells have a negative regulatory function (39). M_reg cells suppress CD4⁺ T cell proliferation by a cell-cell contact-dependent mechanism, and inhibit intestinal inflammation. M_reg cells preferentially associate with CD4⁺ T cells through highly expressed adhesion molecules such as ICAM-1 and VCAM-1, but do not activate CD4⁺ T cells because expression of CD80/CD86 was severely suppressed via IL-10/Stat3 signalling. LysM-cre; Stat3^flox/flox mice, which harbour a Stat3 mutation specifically in myeloid cells, spontaneously develop colitis and show defective M_reg cell function. Transfer of wild-type M_reg cells to Stat3 mutant mice ameliorated intestinal inflammation, suggesting that the dysfunction of M_reg cells is involved in the pathogenesis of intestinal inflammation. However, human counterparts to murine M_reg cells remain elusive.

(iv) Macrophages

Intestinal CD11b⁺CD11c^– macrophages produce large amounts of IL-10 in response to microbiota (40–42). Intestinal macrophage-derived IL-10 inhibits the production of pro-inflammatory cytokines including IL-12 and TNF-α produced by activated intestinal myeloid cells against microbiota by an IL-10/Stat3 signal-dependent mechanism. In addition, IL-10 produced by intestinal macrophages prevents intestinal inflammation by maintaining the persistence of Foxp3 expression in T_reg cells (43). Accordingly, IL-10-deficient mice and LysM-cre; Stat3^flox/flox mice spontaneously develop enteric inflammation accompanied by enhanced effector T cell activity (40, 44).

In patients with CD, numbers of CD14⁺ macrophages are increased and produce greater amounts of colitogenic cytokines such as IL-6, IL-23 and TNF-α against microbiota compared with healthy individuals (45). In addition, CD14⁺ macrophage-derived IL-23 affects the accumulation of colitogenic IL-17 and IFN-γ-producing T cells, suggesting that the abnormal activity of macrophages causes the development and/or pathogenesis of IBD (45–48).

(v) Innate lymphoid cell

In addition to innate myeloid cells, innate lymphoid cells (ILCs) comprising type 1 ILCs (ILC1s), ILC2s, and ILC3s are responsible for immune homeostasis. In particular, RORγt⁺ ILC3s predominantly produce IL-22 and/or IL-17 in the intestine. In both human and mice, IL-23 produced by DCs activates ILC3s responses (49). ILC3s-derived IL-22 promotes production of anti-microbial peptide and mucus production in epithelial cells (50–52), leading to host defence against pathogen such as Citrobacter rodentium (49, 50, 53, 54). In contrast, accumulation of IL-17- and IFN-γ- producing ILC3s is associated with development of colitis during Helicobacter hepaticus infection (55). These results indicate that ILC3s responses induce either host defence or inflammation in accordance with context.

Commensal Bacteria and Gut Homeostasis

The mammalian gastrointestinal tract harbours a huge number of microbial species. Recent studies report that intestinal microbiota mediate the maintenance of gut homeostasis by modulating both nutrient metabolism and host immune responses (56–58) (Fig. 3). Thus, perturbation of the microbiota composition is linked to the pathogenesis of IBD (59–62). Recently, several studies reported that host genetic alterations are implicated in the perturbation of intestinal microbiota composition leading to the development of IBD. Accordingly, IBD patients with NOD2 or ATG16L1 mutations showed altered intestinal microbial composition characterized by decreased amounts of Bacteroidetes and Firmicutes (63).

Fig. 3.

Functions of commensal bacteria on the host immunity. (A) SFB mediate the induction of Th1/Foxp3⁺ T_reg cells and development of Th17 cells in Peyer’s Patches and the small intestine, respectively. (B) Clostridium species initiate the development of Foxp3⁺ T_reg cells in the colon. (C) Bacteroides fragilis-derived polysaccharide A (PSA) contribute to induction of Foxp3⁺ T_reg cells. (D) Micronutrients produced by commensal bacteria including SCFAs and Vitamin 9 participate in maintenance of Foxp3⁺ T_reg cells in the gut.

Th17 Cell Generation by Commensal Bacteria

Segmented filamentous bacteria (SFB) mediate Th17 cell development in the small intestine (64) and induce Th1 cells and Foxp3⁺ T_reg cells in Peyer’s patches (65), indicating that SFB may extensively regulate the intestinal adaptive immune system. Adhesion of SFB induces production of serum amyloid A protein and reactive oxygen species (ROS) in intestinal epithelial cells, leading to induction of antigen-specific Th17 cell development (66). SFB colonization has been reported to protect hosts from C.rodentium infection via the generation of Th17 cells. In contrast, SFB monocolonization of K/BxN mice triggered autoimmune arthritis (67) and enhanced experimental autoimmune encephalomyelitis (68, 69), indicating that the accumulation of Th17 cells by SFB colonization participates in developing autoimmune diseases while contributing to mucosal protection against pathogens.

Foxp3⁺ T_reg cell Generation by Commensal Bacteria

Clostridium species belonging to cluster XIVa and IV initiate the development of Foxp3⁺ T_reg cells in the colon (70). These Clostridium species activates production of matrix metalloproteinase (MMPs) and indoleamine 2, 3-dioxygenase. MMPs generate biologically active TGF-β, leading to the development and maintenance of Foxp3⁺ T_reg cells. In addition, Bacteroides fragilis protect animals from experimental colitis by initiating Foxp3⁺ T_reg cell development (71). The beneficial effect of B.fragilis depends on the expression of polysaccharide A, which is a unique surface polysaccharide that binds to TLR2 on CD4⁺ T cells (72).

Metabolites Derived from Microbiota and Gut Immunity

Micronutrients produced by commensal bacteria, such as short chain fatty acids (SCFAs), lipids and vitamins, can modulate the host immune system and further participate in shaping the microbiota architecture (60, 73). For instance, vitamin B9 also called folic acid derived from diet and commensal bacteria is involved in maintaining immunological homeostasis by promoting the survival of gut Foxp3⁺ T_reg cells with high levels of folate receptor 4 (74, 75). In addition, bile acids synthesized by commensal bacteria suppress the production of IL-6, TNF-α, IL-1β and IFN-γ by inhibiting NF-κB activity via the G protein-coupled bile acid receptor (GPBAR1) and nuclear receptors subfamily 1, group H, member4 (NR1H4, also known as FXR) (76–78) in macrophages. IBD patients show intestinal dysbiosis, which is associated with lower concentrations of bile acid in the faeces and periphery compared with healthy individuals (79), indicating that altered bile acid production by microbiota might contribute to the development of IBD.

SCFAs, such as butyrate, acetate and propionate, are the major metabolites produced by commensal bacteria during fermentation of dietary fibre in the colon. They contribute to the maintenance of gut homeostasis through G protein-coupled receptors (GPRs) including GPR43 and GPR109A (80–83). Acetate produced by Bacteroides thetaiotaomicron facilitates goblet cell development and mucus secretion (84). Bifidobacterium longum can suppress the translocation of E. coli O157:H7 Shiga toxin by promoting intestinal epithelial integrity through the production of acetate (85). In addition, acetate promotes the development of Th1 and Th17 cells by inhibiting histone deacetylases (HDACs) activity that regulates the mTOR pathway (86). In contrast, butyrate and propionate promote the accumulation of Foxp3⁺ T_reg cells and their suppressive activity by inhibiting HDAC activity through GPR43 (87, 88). Butyrate also negatively regulates the production of pro-inflammatory cytokines including IL-12, IL-6, TNF-α, and nitric oxide from macrophages through HDAC inhibition (89, 90). Accordingly, HDAC inhibitors have potential therapeutic benefit for IBD.

Conclusion

Recent advances have revealed the crucial roles of innate immune cells for immunological tolerance and protection against invading pathogens. Abnormal activity of innate immunity in the intestinal lamina propria can lead to the development of IBD by inducing inappropriate Th1/Th17 responses whereas normal Th1 and Th17 responses protect against pathogens for host defence. Thus, Foxp3⁺ T_reg cells, which abundantly exist in the intestinal mucosa, inhibit innate and adaptive immune responses to prevent intestinal inflammation. In addition, recent studies showed that microbiota and nutritional metabolites derived from commensal bacteria are implicated in the maintenance of gut homeostasis and an imbalance in the composition of gut microbiota influences the pathogenesis of IBD. Thus, further studies to characterize the mechanisms by which commensal bacteria regulate the innate/adaptive immune systems may promote advances in therapeutic approaches for IBD.

Glossary

Abbreviation

CCR9

CC chemokine receptor 9

CX₃CR1

CX3C chemokine receptor 1, Foxp3, Forkhead box P3

HDAC

histone deacetylase

Funding

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology; the Japan Science and Technology Agency, and by the Ministry of Health, Labour, and Welfare.

Conflict of Interest

None declared.