Immune responses of TLR5+ lamina propria dendritic cells in enterobacterial infection
Toll-like receptors (TLRs) recognize distinct microbial components and induce innate immune responses. TLR5 has been shown to recognize bacterial flagellin. Unlike other TLRs, TLR5 is not expressed on conventional dendritic cells or macrophages. By contrast, TLR5 is mainly expressed on intestinal CD11c+ lamina propria cells (LPCs), which do not express TLR4. These cells detect pathogenic bacteria and secreted proinflammatory cytokines, mainly in a TLR5-dependent manner. Notably, transport of pathogenic Salmonella typhimurium from the intestinal tract to mesenteric lymph nodes was impaired in Tlr5 −/− mice, suggesting that CD11c+ LPCs expressing TLR5 are used by S. typhimurium for systemic infection. CD11c+ LPCs consist of four subsets distinguished by differential expression patterns of CD11c and CD11b. CD11chiCD11bhi LPDCs have been identified as TLR5-expressing cells. In response to flagellin, these LPDCs induce the differentiation of naive B cells into IgA+ plasma cells via a mechanism independent of gut-associated lymphoid tissue (GALT), and trigger the differentiation of antigen-specific Th17 and Th1 cells. These LPDCs have unique properties in that they can induce acquired immunity as well as innate immunity via TLR5 in the intestine.
KeywordsLamina propria TLR5 IgA Th17 Autophagy
Toll-like receptors (TLRs) recognize a variety of microbial components and induce innate immune responses . They are abundantly expressed on ‘professional’ antigen-presenting cells (APCs) such as macrophages and conventional dendritic cells (DCs) and serve as an important link between the innate and adaptive immune responses. To date, 13 TLRs have been identified in mammals. Bacterial flagellin is a structural protein that forms the main portion of flagella and promotes bacterial chemotaxis and bacterial adhesion to and invasion of host tissues . TLR5 recognizes the conserved domain in flagellin monomers and triggers proinflammatory as well as adaptive immune responses [3, 4]. TLR5 is expressed on the basolateral surfaces of intestinal epithelial cells and is thought to be key in the recognition of invasive flagellated bacteria at the mucosal surface . Human intestinal epithelial cell lines produce chemokines in response to flagellin, leading to subsequent migration of immature DCs . Furthermore, TLR5 is highly expressed in the human lung and a common TLR5 polymorphism in humans causes susceptibility to legionellosis [7, 8]. Although accumulating evidence suggests that TLR5 is critical for host defense against enterobacterial infection, the in vivo function of TLR5 has not been fully elucidated.
Identification of TLR5-expressing cells in the intestine
Innate immune responses by CD11c+ LPCs
Because the intestine is constantly exposed to food antigens (Ags) and commensal bacteria, it is the consensus that most mucosal DCs induce tolerance, but not inflammation. In the Peyer’s patches and mesenteric lymph nodes, conventional DCs consist of CD11chiCD11b+CD8a–, CD11chiCD11b–CD8a+ and CD11chiCD11b−CD8a− subsets. In addition, CD11cint plasmacytoid DCs exist in these sites . DCs in the Peyer’s patch produce interleukin 10 (IL-10) rather than IL-12, polarize naive T cells toward T helper type 2 (Th2) or regulatory T (Treg) cells, and induce the differentiation of immunoglobulin A (IgA)+ plasma cells . Flagellin-stimulated CD11c+ LPCs do not produce IL-10 and tumor-necrosis factor (TNF)-α, but instead produce IL-6 and IL-12 . Furthermore, CD11c+ LPCs express chemokines, prostaglandins, antimicrobial peptides and molecules involved in cellular adhesion, cytoskeletal organization and intracellular transport in response to flagellin . Accordingly, CD11c+ LPCs have a tendency to induce inflammatory responses rather than tolerance when exposed to flagellin.
Recognition of bacteria by CD11c+ LPCs
TLR4, which is abundantly expressed on innate immune cells such as conventional DCs and macrophages, recognizes lipopolysaccharide (LPS) of Gram-negative bacteria . However, CD11c+ LPCs do not produce any inflammatory cytokines in response to LPS owing to their low expression of TLR . Conventional DCs, which express TLR4 but not TLR5, recognize Gram-negative flagellated bacteria, mainly via TLR4, while TLR4−TLR5+CD11c+ LPCs produce inflammatory cytokines after exposure to such bacteria . The unique profile of TLR expression in CD11c+ LPCs seems to be closely related to the specific environment in the intestine. Most commensal bacteria in the intestine are Gram-negative anaerobic rod bacteria, which contain LPS in their cell walls. The low expression of TLR4 may allow CD11c+ LPCs to avoid inducing excessive immune responses to commensal bacteria. Instead, CD11c+ LPCs may induce inflammatory responses to pathogenic flagellated bacteria, which are able to invade the LP, via TLR5. However, some commensal bacteria also have flagella. Recently, it was reported that α- and ε-proteobacteria can change the TLR5 recognition site in flagellin without losing flagellar motility . Furthermore, some commensal bacteria in the intestine suppress flagellin expression . Unlike pathogenic bacteria, commensal bacteria may have mechanisms to escape TLR5-mediated host detection in the intestine.
Role of TLR5 in Salmonella typhimurium infection
Four subsets of CD11c+ LPCs in the small intestine
TLR5 and intestinal IgA production
The intestine is known as an organ that produces large amounts of secretory IgA . Intestinal gut-associated lymphoid tissues (GALTs) such as PPs, ILFs and MLNs are major sites for generation of IgA+ plasma cells in the intestine. IgA+ plasma cells are induced there via a mechanism dependent on Ag, CD4+ follicular B helper T cells and the formation of germinal centers (GCs) [20, 21, 22]. Suppressive Foxp3+CD4+ T cells in PPs can differentiate efficiently into cells with characteristics of follicular B helper T cells, which then participate in the induction of GCs and IgA production in the gut. CD103+ GALT DCs produce retinoic acid (RA), which induces the selective expression of gut homing receptors, such as integrin α4β7 and CCR9, on differentiated IgA+ plasma cells for gut homing [24, 25]. However, differentiation of IgA+ cells does not necessarily require T cell help and GC formation. GALT DC-derived RA and cytokines synergistically act on naïve B cells, leading to the generation of T cell–independent IgA+ cells [25, 26]. Furthermore, some IgM+ B cells, especially peritoneal B1 cells, directly migrate to the gut LP in a sphingosine 1-phosphate (S1P)-dependent manner and differentiate into IgA+ plasma cells in the LP with the help of stroma cells . Commensal bacteria induce natural secretory IgA, and this process is mediated by commensal bacteria-laden DCs . Furthermore, DCs in the small intestinal LP send protrusions into the lumen through an intercellular gap between epithelial cells via a mechanism dependent on the chemokine receptor CX3CR1, and actively sample luminal bacteria . Although accumulating evidence suggests the involvement of DCs in the generation of IgA+ plasma cells in the LP, it is not known what subset of DCs is responsible for this event. Interestingly, CD11chiCD11bhi LPDCs are responsible for the generation of IgA+ plasma cells in the LP. Flagellin-stimulated CD11chiCD11bhi LPDCs efficiently induced the differentiation of B220− IgA+ plasma cells in the absence of T cells in a TLR5-dependent manner. Similar to CD103+ DCs in GALTs, CD11chiCD11bhi LPDCs specifically express Aldh1a2 mRNA, which encodes retinal dehydrogenase 2 (RALDH2). CD11chiCD11bhi LPDC-derived RA synergistically acts on naïve B cells with TLR5-mediated inflammatory cytokines, leading to the induction of IgA+ plasma cells. Furthermore, TLR5 is critical for IgA synthesis in vivo. Mice lacking the transcription factor Id2 do not develop GALTs, yet they retain intestinal IgA production. In wild-type mice, around 20% of IgA+ plasma cells exist in the small intestinal LP. Interestingly, about 10% of IgA+ cells are found in the LP of Id2 −/− mice, which confirms that gut IgA can be generated without GALTs. Although Tlr5 −/− mice did not have fewer IgA+ B cells, Id2 −/− Tlr5 −/− mice had far fewer IgA+ cells in the LP . Thus, TLR5 signaling in CD11chiCD11bhi LPDCs is essential for GALT-independent IgA synthesis.
CD11chiCD11bhi LPDC-mediated CD4+ helper T cell (Th) response
A series of recent studies showed that RA negatively regulates Th17 cell differentiation [47, 48, 49]. In every study, RA effectively inhibited in vitro differentiation of Th17 cells induced by IL-6 plus TGF-β in a dose-dependent manner. Although CD11chiCD11bhi LPDCs express RALDH2, they specifically induce Th17 cells . Interestingly, the effect of RA on DC-mediated Th17 cell differentiation differs according to its concentration. RA at high concentration (10 μM) inhibits the differentiation of Th17 cells as well as Th1 cells, suggesting that high concentrations of RA inhibit both Th17 and Th1 cell differentiation induced by LPDCs. On the other hand, the RAR inhibitor LE540 inhibits the differentiation of Th17 cells, but not Th1 cells, suggesting that RA from LPDCs is necessary for Th17 cell differentiation. Interestingly, LPS-stimulated SPDCs can induce Th17 cells to the same extent as flagellin-stimulated LPDCs (R2) following the addition of RA at low concentration (1 nM). Furthermore, 10 μM RA abolished Th1 cell differentiation induced by LPS-stimulated SPDCs . Thus, RA at low concentrations (1 nM) may have a positive effect on DC-mediated Th17 cell differentiation. According to a previous report, the plasma RA level is usually on the order of 10 nM . It is well known that GALT DCs, but not DCs from other central lymphoid tissues, such as the spleen or peripheral lymph nodes (PLNs), produce RA, which upregulates expression of the chemokine receptor CCR9 and the integrin α4β7 on CD8+ precursors, Ag-specific CTLs, CD4+ T cells (Th1 and Th 2) and B cells [24, 25, 50, 51, 52]. Iwata et al  clearly showed that RA effectively enhanced α4β7 expression on CD8+ T cells even at concentrations in the range 0.1–1 nM. Because CD11chiCD11bhi LPDCs could induce B220−IgA+ plasma cells from naïve B cells and CCR9 expression on differentiated plasma cells effectively in vitro, the RA released from LPDCs is functional . Although it is difficult to determine the local concentrations of retinoic acid secreted by CD11chiCD11bhi LPDCs, LPDC-derived RA acts as a positive regulator of Th17 cell differentiation. Thus, we have to reconsider the effects of RA on Th17 responses more cautiously.
CD11chiCD11bhi LPDCs and regulatory T cells
Recent reports show that CD103+ DCs in MLNs and LPs have a role in regulating immunity [53, 54]. CD103+ DCs migrate from the LP to MLNs in a CCR7-dependent manner and induce FoxP3+ regulatory T (Treg) cells via their derived RA [55, 56]. CD103+ DCs seem to be the same population as CD11chiCD11bhi LPDCs, because CD11chiCD11bhi LPDCs specifically express CD103 and RALDH2 . CD11chiCD11bhi LPDCs may have opposite functions, inducing both immunological tolerance and protective immune responses. When CD11chiCD11bhi LPDCs take up food antigens under physiological conditions, they may induce immunological tolerance by promoting the development of T reg cells. When CD11chiCD11bhi DCs take up pathogenic bacteria during infection, they are activated and induced to maturate via innate immune receptors, leading to the activation of acquired immunity through the induction of proinflammatory cytokines and costimulatory molecules. Thus, CD11chiCD11bhi DCs may play critical roles in the decision to mount tolerant or protective immune responses in intestine by using innate immune receptors such as TLR5 as switches.
We thank our colleagues in our lab for helpful discussion and E. Kamada for secretarial assistance. This work is supported in part by grants from the Special Coordination Funds of the Japanese Ministry of Education, Culture, Sports, Science and Technology.