Abstract

Introduction

In mucosal tissues, the development of inflammation and immunity is central to effective host defense against invading pathogens, yet must be tightly regulated to prevent abnormal responses to innocuous environmental antigens and commensal organisms that result in allergy or chronic inflammatory diseases. This is particularly true in the intestine, where there is continuous exposure to a vastly complex mixture of ingested environmental and food antigens, and intestinal microbiota.

This review will highlight recent findings regarding the phenotype and function of dendritic cells (DCs) and macrophages in the induction and regulation of immune responses in the intestine. DCs are likely key players in regulating immune responses in the intestine since they are prominently localized to mucosal surfaces, both at sites of antigen uptake and within inductive lymphoid tissue, have been shown to process antigens given in both a tolerogenic and immunogenic form, and to directly sample endogenous flora and pathogenic microorganisms in vivo. Furthermore, subpopulations of mucosal DCs have been identified that have unique functions when compared to DCs from non-mucosal sites, including the imprinting of mucosal homing receptors on T and B cells, the induction of regulatory T cells to soluble antigens in the resting, or “steady” state, and the direct contribution to IgA B cell class switching. Mucosal macrophages are also found prominently in the intestine, primarily within the lamina propria, and have been shown particularly capable of taking up and killing bacteria. Recent evidence in the mouse also suggests that intestinal macrophages have unique immunoregulatory roles including the production of suppressive cytokines and the ability to induce the differentiation of regulatory T cells.

Antigen uptake and cell trafficking

Important for understanding how mucosal immune responses are induced and regulated is the issue of where different types of antigens are processed and presented to T and B cells. Primary sites for the induction of intestinal T and B cell responses are Peyer’s patches (PPs) in the small intestine, isolated lymphoid follicles (ILF) in the small and large intestine, and mesenteric lymph nodes (MLN). In contrast, the diffuse lamina propria (LP) and the intraepithelial cell (IEC) compartments are primarily effector sites.

Luminal antigens including macromolecules, bacteria, and viruses, gain access to the cells of PPs and ILF via specialized, antigen-transporting epithelial cells, M (micro-fold) cells, present in the follicle-associated epithelium (FAE) above organized lymphoid structures of most mucosal tissues^1,2. M cells transport is promiscuous and mediated by binding to surface expressed carbohydrates in regions free of overlying mucus, but can be enhanced by the presence of antigen-specific IgA^3–5, by immune targeting with anti-M-cell antibodies⁶, or by oral administration of TLR2 or TLR4 ligands^7,8.

DCs are present within the FAE in small numbers and in large numbers the SED⁹. Furthermore, orally administered cholera toxin, cholera toxin B-subunit, or E. coli heat-labile toxin (LT)¹⁰, and proteosome vaccines⁸ as well as Salmonella Typhimurium infection¹¹ can induce an influx of DCs from the SED into the FAE. In the latter study, PP DCs in the SED expressing CCR6 appear to migrate into the FAE, where they form clusters with antigen-specific CD4 T cells, and the activation and expansion of specific T cells was dependent on CCR6¹¹. In contrast, CCR6-expressing DCs were not recruited to the intestinal lamina propria, suggesting that CCD6+ DCs may have specific functions in organized lymphoid structures, where the CCR6-ligand, CCL20 is constitutively expressed¹².

PP DCs in the SED capture soluble antigens given orally^9,13, and take up, or are initial targets of orally administered pathogens, including S. typhimurium^14,15, Listeria monocytogenes¹⁶, Brucella abortus¹⁷, and H. pylori¹⁸. Furthermore, PP DCs in the SED take up apoptotic epithelial cells following intestinal reovirus infection¹⁹.

Following activation DCs in the SED can migrate to T cell zones, as shown following oral CT or systemic administration of a soluble antigen preparation from Toxoplasma gondii tachyzoites (STAg)¹². In the later study, PP DCs down regulated CCR6, expressed CCR7 and migrated to T cell zones where CCR7-ligands CCL19 and CCL21 are expressed¹².

Increasing data also suggests that PPs are primary sites for the uptake of commensal bacteria, and that DCs may be primary targets. Following oral administration of Enterobacter cloacae, organisms were found in DCs in the PPs and MLNs, but not in the LP or spleen, where they likely help drive IgA production that then limits their penetration²⁰. Organisms were not found in presumed macrophages (CD11b+ CD11c− cells) from these tissues, and in contrast to DCs, ex vivo CD11b+CD11c− cells were able to kill the commensal bacteria efficiently, indicating that commensal bacteria can be taken up and persist in DCs in PPs (and possibly ILFs) and MLNs, but cannot migrate to systemic sites, thus limiting their potential to cause systemic inflammation²⁰. Furthermore, specific IgA induced against commensal bacteria may target these for further uptake into PPs and ILFs⁵. Interestingly, initial erosions in ileal Crohn’s disease appear to occur over lymphoid follicles, and uptake of non-pathogenic E. coli by FAE is enhanced in patients with long-standing disease, suggesting that abnormal uptake and/or poorly regulated immune responses to commensal bacteria in PPs may play a role in early disease pathogenesis^21,22.

The extent to which PP DCs traffic to the MLN is this or other situations is not yet clear, however, the presence of chemokines important for DC migration to LN, such as CCL19, and CCL21 within the T cell zones of PPs¹² suggests that PP DC migration, as well as specific immune responses, may be relatively contained within the PP. Furthermore, phenotypic analysis of DC subpopulations in the rat indicate that the primary source of migratory DCs is the intestinal LP and not the PPs²³.

A second site for antigen entry into the intestine is the non-follicular absorptive epithelium, where both soluble antigens and bacteria can gain access to DCs in the lamina propria (LP). This can occur by trans- or para-cellular transport, by receptor mediated trafficking, such as occurs via the neonatal FcR expressed on absorptive epithelial cells in humans²⁴, by directly sampling of luminal contents by DC extensions that reach between epithelial cells into the intestinal lumen^25–27, or by direct damage to the epithelium, as can occur during inflammatory bowel disease, or infection with HIV²⁸ or Shigella flexneri²⁹. Antigen sampling may also occur by uptake of exosomes from epithelial cells³⁰

Even in the absence of infection or inflammation, LP DCs consitutively traffic to MLNs³¹ which appears to be a relatively active process. These migratory DCs can carry self- or cell-associated antigens from apoptotic epithelial cells³² or soluble proteins given orally^33–35. Soluble antigens given orally can be processed by LP DCs, which then migrate to the MLN in a CCR7-dependent manner, which was shown to be essential for oral tolerance induction³⁵. In addition, commensal bacteria are present within LP DCs, and both S. typhimurium and non-invasive E. coli can be taken up by luminal dendrites in the terminal ileum when given orally^25,26, which may carry these bacteria to the MLN.

LP DCs can actively take up pathogenic bacteria. Whereas DC uptake of non-invasive bacteria appears to occur predominantly via DC extensions into the intestinal lumen, a process that is dependent on CX3CL1-CX3CR1 interactions²⁶. Interestingly, it was recently shown that the major route of entry into the body for non-invasive S. typhimurium may indeed be via DC extensions, while pathogenic S. typhimurium preferentially invades PPs to induce systemic pr mucosal humoral immune responses, respectively³⁶. Activation of LP DCs results in enhanced migration to MLNs, as occurs following systemic administration of LPS, or orally administered TLR7/8 agonists^37,38. Furthermore, when compared to DCs from other sites, LP DCs preferentially express TLR5³⁹, which upon activation by bacterial flagellin⁴⁰, may be important for DC activation and migration to MLNs, or spread of invasive bacteria to systemic sites³⁹. These studies indicate that LP DCs are fully capable of becoming activated by microbial or inflammatory products, resulting in enhanced migration to MLN.

The expanding family of intestinal DC populations and their specialized functions

Multiple DC subpopulations have been identified in the PPs, MLN, and intestinal lamina propria, which differ in their surface phenotype, localization, cytokine production and ability to drive T cell differentiation in vitro. These studies have been the subject of several recent reviews^41–44 and see Table 1. Over the past several years, several unique functions of intestinal DCs have been identified including the imprinting of lymphocytes with unique homing receptors that allow for their recirculation to intestinal tissues, the capacity to provide direct signals for the differentiation of IgA-producing B cells, and the ability to drive the differentiation of regulatory T cells that are involved in tolerance to soluble oral antigens and commensal bacteria.

Induction of homing receptors

Initial studies implicating the importance of the lymphoid microenvironment in the imprinting of homing receptors on T cells demonstrated that adoptively transferred TCR-transgenic CD4+ and CD8+ T cells primed in the mesenteric, but not cutaneous LNs or spleen expressed high levels of α4β7, an integrin that binds to MadCAM-1 expressed on high-endothelial venules of intestinal tissues, and CCR9 and migrated in response to CCL25, the chemokine ligand of CCR9 expressed constitutively in the small intestine^45–47. Subsequently, it was shown that T cells primed in vitro with antigen-pulsed MLN or PP DCs, but not DCs from the spleen or peripheral lymph nodes (PLN) expressed CCR9 and high levels of α4β7^46,48,49. Furthermore, PP DC-primed CD8+ T cells had enhanced migration to the small intestine⁴⁸ which was dependent on a fixation-sensitive signal from the DCs⁵⁰.

The capacity of intestinal DCs to drive intestinal homing receptors on T cells is largely dependent on retinoic acid (RA) a metabolite of retinol (vitamin A). Dietary retinol, or retinoids hydrolyzed to retinol, are stored in the liver and released at a constant level in the blood. Retinol becomes successively oxidized inside cells to retinal by alcohol dehydrogenases (ADH), or members of the short-chain dehydrogenase/reductase (SDR) family, and then to RA by retinal dehydrogenases (RALDH). RA signaling in mediated by nuclear receptors of the retinoic acid receptor (RAR) and retinoid X (RXR) families, which form RAR/RXR heterodimers. Upon RA binding to the RAR, the RAR/RXR heterodimers act as transcription factors.

In initial studies, exogenous RA directly drove the expression of α4β7 and CCR9 on T cells activated in vitro with anti-CD3 and anti-CD28, which homed to the intestine. Furthermore, PP and MLN DCs expressed RALDH enzymes, produced RA from retinol, and inhibitors of RA production and signaling blocked DC-induced α4β7 and CCR9 expression. Finally, mice on vitamin A-deficient diets had a reduction in α4β7⁺ memory T cells in lymphoid organs and a dramatic deficiency of LP T cells in the small intestine⁵¹. Interestingly, epithelial cells in PPs also expressed RALDH1, indicating that RA production is unlikely restricted to DCs. In addition, it is possible that RA from epithelial cells conditions local DCs for the ability to produce TGF-beta and IL-6, and the capacity to augment mucosal homing receptor expression⁵².

In studies from 2 laboratories, DCs expressing αEβ7 integrin (CD103) in the intestine were shown to have unique functional properties^53,54. In studies of homing receptor expression, LP DCs were demonstrated to be as potent as MLN DCs in inducing α4β7 on CD8⁺ T cells, and better at inducing CCR9. Extensive phenotypic analysis identified particularly high numbers of DCs expressing CD103 in the LP compared with the MLN⁵⁴. Furthermore, CD103⁺ DCs from the MLN were found to express higher levels of MHC II than the same cells from the LP, and to be far lower in number in the MLN of mice lacking CCR7, indicating that this population likely migrates from the LP to the MLN, and represents in the mouse, the “semimature” DCs that were found migrating in the steady state in intestinal lymph in the rat. When tested in vitro, only CD103+ DCs from either the MLN or LP were found to drive the expression of high levels of CCR9 and a4b7 on CD8 T cells. Furthermore, CCR9 and a4b7 were not induced on CD8+ T cells in MLNs of CCR7−/− mice given systemic antigen and LPS, despite equivalent levels of proliferation of the CD8+ T cells in the MLN.

In studies in the T cell transfer model of colitis, CD103 expression by host-cells was essential for the ability of CD4+CD25+ regulatory T cells to protect against colits induction⁵³. DCs were the predominant host cell expressing CD103 in the spleen, and high numbers of CD103+ DCs were found in the colon and MLN. Interesingly, CD103 expression was found on the 3 previously identified DC subsets from the spleen, MLN and colon, those that were CD8+/CD11b−, CD8−/CD11b+, and CD8−/CD11b−, with the highest percentage of CD103⁺ DCs in the CD8⁺ population. Functional studies demonstrated that CD103+ DCs, but not their CD103− counterparts, promoted expression of the gut-homing receptor CCR9 on CD4⁺ T cells. Collectively, these studies indicated that a subpopulation of MLN DCs expressing CD103 were likely derived from the intestinal LP and upon migration to MLNs were essential for driving homing receptors on CD4⁺ and CD8⁺ T cells.

Most recently, studies using RA-responsive element reporter mice demonstrated that while both spleen and MLN DCs were capable of driving RA-receptor (RAR) signaling and α4β7 expression in CD8+ T cells, only CD103+ DCs from the MLN drove an early RAR signal that was required for CCR9 and high levels of α4β7 expression in vitro⁵⁵. Furthermore, CD8+ T cells primed in vivo in the MLN had evidence of enhanced RAR signaling. Interestingly, DC-mediated induction of gut homing receptors was inhibited on CD8+ T cells at a high antigen dose without influencing RAR signaling events, indicating that the induction of gut-homing receptors is likely controlled by both the intensity of RAR signaling, as well as antigen dose. These data implied that the early and high levels of RA production by mucosal CD103⁺ DCs is largely responsible for their ability to drive CCR9 and α4β7 expression on activated lymphocytes, and that this can be overcome by high antigen doses.

Finally, similar to what was shown for T cells, PP and MLN DCs induced CCR9 and α4β7 expression on both naïve and antigen experienced B cells stimulated with anti-IgM that was dependent on RA, and allowed B cells to selectively home to the intestine⁵⁶. Furthermore, repeated stimulation of human naïve B cells with spleen DCs +/− RA, demonstrated flexibility in home receptor expression, in that cells initially stimulated in the absence of RA, still expressed high levels of CCR9 and α4β7 expression on repeated activation in the presence of RA, and visa versa. This implicated GALT DCs in driving homing receptors on both T and B lymphocytes, that was dependent on RA.

Induction of IgA responses

A central function of the mucosal immune system is the production of IgA. High affinity IgA acts to exclude microorganisms and toxins from entering the body, while low-affinity IgA is thought to inhibit the binding of commensal bacteria to epithelial cells, the latter of which is thought to be important in the maintenance of an appropriate intestinal microbiota⁵⁷. Naïve B cells are induced to undergo class switch recombination (CSR) and somatic hypermutation in organized lymphoid tissues of the intestine, however recent data indicate CSR can also occur in the diffuse LP, where activation-induced cytodine deaminase (AID), an essential enzyme for isotype-switiching has been detected in B cells (see⁵⁷). In organized tissues, such as the PP, MLNs and ILFs, local factors, including TGFβ, IL-6, and IL-10, together with activation of CD40 by T cell-expressed CD40L, which drives AID expression, are thought to be responsible for T cell dependent IgA B cell differentiation. In the LP, TLR-dependent induction of innate IgA CSR-inducing factors, including APRIL and its homologue B-cell-activating factor of the tumor necrosis factor family (BAFF, also known as BLyS) from epithelial cells, DCs and B cells are thought to drive T cell independent IgA B cell differentiation.

Recent studies have implicated a unique role for mucosal DCs in regulating IgA B cell differentiation. First, bacteria are present in lymphoid follicles, including Peyer’s patches, and the lamina propria in the terminal ileum of normal mice, and can be found within DCs^20,58–60. In addition, both humans and mice produce significant amounts of secretory IgA against commensal bacteria, which is induced in the absence of T cells²⁰.

Second, PP DCs were recently shown to not only induce homing receptors on B cells (as noted above, but also to drive IgA B cell differentiation from naïve B cells in the absence of T cell signals via the production of RA and IL-6⁵⁶. Furthermore, DCs from peripheral LNs or the liver, were capable of inducing IgA B cell differentiation in the presence of exogenous RA and either IL-5 or IL-6, but, interestingly, not TGFβ. Finally, Vitamin A-deficient mice, had a paucity of IgA+ B in the LP, but normal numbers of naïve, IgM+ B cells in PPs⁵⁶. Therefore, mucosal DCs can contribute to direct IgA B cell differention to conmmensal bacteria, either in lymphoid structures or in the LP via their ability to produce RA and TLR-induced IL-6, and possibly BLyS/April. Furthermore, their ability to produce TGFβ, as well as their superior capacity to activate naïve T cells most certainly indicate a central role for DCs in T cell-dependent IgA B cell differentiation in organized lymphoid structures. In support of this possibility, CD11b+ DCs from PPs were shown to be superior to other DC populations in driving IgA B cell differentiation in the presence of cognate T cells in vitro, a function dependent on their ability to produce IL-6⁶¹.

Oral tolerance and the peripheral differentiation of regulatory T cells

Several mechanisms have been identified by which systemic tolerance to orally administered antigens (oral tolerance) is induced, which have been largely identified in mice fed soluble proteins, such as ovalbumin, myelin basic protein (MBP), retinal S-antigen, collagen, and insulin, as well as peptides^62,63. These mechanisms include the induction of T cell anergy, deletion, and the induction of CD4⁺ Tregs. The mechanisms involved are influenced by antigen dose and frequency with higher doses favoring anergy and deletion, and lower and repeated doses the generation of regulatory T cells capable of transferring to naïve mice tolerance to subsequent immunization or the induction of autoimmune disease⁶². The induction of antigen-specific Tregs capable of bystander suppression, were initially identified as Th3 cells producing TGFβ⁶⁴. Later studies demonstrated that oral antigen administration could also induce the differentiation or expansion of antigen-specific CD4+CD25+ Tregs in the MLN that could transfer tolerance to naïve mice^65,66. Furthermore, naturally occurring CD4+ CD25+ T cells were required for oral tolerance in a CD8+ T cell-dependent model of skin contact hypersensitivity⁶⁷.

Initial studies implicating DCs in oral tolerance demonstrated that expansion of DCs in vivo with Flt3L administration resulted in an enhanced sensitivity to oral tolerance induction⁶⁸. In addition, PP DCs could be loaded with soluble oral antigens^9,13, and DCs from the PPs but not the spleen were shown to produce IL-10, and likely TGFβ⁶⁹, and both PP DCs⁶⁹ and MLN DCs from fed mice⁷⁰ were able to drive the differentiation of T cells producing IL-4 and IL-10 in vitro. Furthermore, PP CD11b⁺ DCs were unique in their ability to produce IL-10 and drive the differentiation of IL-10- and IL-4-producing T cells⁷¹. Finally, DCs from the LP were shown to take up oral antigens, to express mRNA for IL-10 and IFN-γ, but not IL-12, and following antigen feeding were able to induce tolerance when transferred to naïve mice⁷². Together, these studies indicate that PP and LP DCs can drive non-inflamatory T cell responses tolerance following antigen feeding.

Recently, significant new studies have emerged supporting the concept that mucosal DCs are indeed different in their ability to drive T cell responses, in particular in their ability to drive de novo induction of CD4+Foxp3+ Tregs^73–75. CD4+ Foxp3+ Tregs can differentiate in the thymus in response to self-antigens expressed at this site during a restricted period of post-natal development⁷⁶. In addition, CD4+FoxP3+ Tregs can differentiate from CD4+ CD25− T cells in vitro in the presence of TGFβ^77–79, which are functionally relevant as they can suppress experimental colitis induction⁸⁰. Most recently, peripheral induction of Foxp3+ Tregs was found to occur primarily in lymphoid tissues associated with the intestine (Peyer’s patches, mesenteric lymph nodes, and small intestinal lamina propria),^73,81. Importantly, following adoptive transfer of naïve TCR-transgenic T cells to normal mice, oral antigen feeding resulted in accumulation of antigen-specific Foxp3+ Tregs in the PP, MLN, and small intestinal LP⁸¹, indicating a role of peripheral Treg induction in oral tolerance.

Interestingly, DCs from mucosal tissues (MLN or small intestine lamina propria), were more capable than spleen DCs of inducing Foxp3 expression in the presence of exogenous TGFβ^74,81,82, and MLN DCs were less capable of inducing Th17 cells in the presence of TGFβ and IL-6⁷⁴. Furthermore, CD103+ DCs but not CD103− DCs from the small intestinal LP or MLN were shown to induce the differentiation of Tregs in the absence of exogenous cytokines^73,81. This occurred by their production of retinoic acid (RA) and TGFβ^73,81. The induced Foxp3+ Tregs were as efficient as naturally occurring Tregs in suppression assays in vitro and in vivo.

RA also enhanced the in vitro generation of Foxp3+ Tregs from naive T cells in the presence of IL-2 and TGFβ in the absence of DCs^83,84. Furthermore, the addition TGFβ and RA, but not RA alone to spleen or CD103⁻ MLN DCs was able to significantly enhance the differentiation of Foxp3+ Tregs, while TGFb alone has modest enhancing effects, indicating that RA likely acts to enhance TGFβ-mediated Treg differentiation^73,74,81.

In addition to positive effects on Treg differentiation, RA was able to suppress Th17 induction in the presence of IL-6 and TGFβ^74,83; and IL-6 capable of inhibiting Foxp3 induction with TGFβ and RA, depending on the doses of RA and IL-6 in the cultures⁷⁴. This suggests that IL-6 (and possibly other cytokines, e.g., IL-1, TNFα) act in a reciprocal fashion with RA to control Treg induction in the presence of TGFβ-rich environment of the intestine.

In contrast to CD103+ DCs, CD103− DCs induced Th1 differentiation, and produced significant amounts of IL-6, TNFα and IL-23 in response to LPS or CD40 signaling, while CD103+ DCs were much less responsive to stimulation, and were shown to contain enzymes (aldh1a2) involved in conversion of retinal to RA⁷³. In contrast, both CD103+ and CD103− DCs in the intestinal LP were capable of driving nearly equivalent Treg differentiation in the presence of TGFβ that was dependent on RA⁸¹. Therefore, consistent with studies of intestinal homing recepeptor expression, induction, it appears that CD103+ DCs in the MLN are derived from the intestinal LP, while the MLN CD103− DCs may have come directly from blood precursors⁷³, as well as that the LP and not the MLN microenvironment may be important for DC conditioning for Treg induction.

Intestinal Macrophages

While both members of the mononuclear phagocyte system, DCs can be distinguished from macrophges based on their dendritic morphology, their ability to capture, process and present antigens to naïve T cells, and their unique life cycle, acting as sentinels in peripheral tissues that upon tissue or microbial signals carry self and foreign antigens to draining lymphoid tissues for the induction of T cell tolerance and immunity. As such, DCs have developed unique endocytic system for processing of antigens, are localized in many peripheral sites exposed to the external environment, and go through a defined process of maturation in response to a variety of stimuli. Macrophages belong to a vast family of tissue cells, including Kupffer cells in the liver, and glial cells in the brain, but generally share similar several general functional attributes, at least in many tissues in which they are found. In particular they have predominantly innate immune functions, such as the capture and killing of microbes, the scavenging of apoptotic and dead cells, and the production of regulatory cytokines, and are less efficient at presenting antigen to T cells. What is clear, however, is that the phenotype and functions of both DCs and macrophages can vary depending on the tissue and the presence of tissue injury, inflammation, and exposure to microorganisms and external antigens. In addition, there is significant overlap in the origins, and surface characteristics and many functional attributes of these cell types, in particular as more subpopulations of these cell types are defined.

Macrophages have been defined in the intestinal tract in both mice and humans, and are present in high numbers. In fact, in early studies, based on the expression levels of the F4/80 glycoprotein, which is present on circulating monocytes as well the vast majority of tissue macrophages in the mouse, the small and large intestines contained by far the largest reservoir of these cells in the body⁸⁵. F4/80-positive cells are found extensively in the small and large intestine, where they are in close contact with the epithelium, and express CD11b and low to modest levels of MHC II^86,87. F4/80+ cells have also been identified in PPs of mice, however at very low numbers and are found at the follicle base near the draining the lacteals⁹. In other species, including humans, highly phagocytic cells described as macrophages, containing bacteria are also found in the SED of the PP. In humans LP macrophages in the colon have been defined to express CD11b, and low levels of MHC II, but also express microsialin (CD68), and low levels of CD11c⁸⁸, and in the small intestine, macrophages express high levels of MHC II and CD13, a zinc metalloproteinase, but negligible levels CD11b and CD11c⁸⁹.

Although based primarily on human studies, intestinal macrophages from humans and mice appear to share several unique characteristics in the steady-state. First, intestinal macrophages avidly phagocytose particulate antigens and bacteria, and despite their lack of respiratory burst capacity, are highly active in killing these organisms^20,89. Second, they are highly suppressed in their responses to activating signals from cytokines or pathogens, including TLR ligands, that typically induce the production of pro-inflammatory mediators and cytokines and enhance the antigen-presenting capacity of monocytes. This includes the poor cytokine and chemokine production, as well as the lack of inducible expression of co-stimulatory molecules. Intestinal macrophages lack or have poor expression of most innate response receptors, notably CD14, Fc and Fc receptors, TLR1-5 and TREM-1 and (^{90 91}), or have suppressed NFkB-signaling, possibly due to the presence of suppressive cytokines, such as IL-10 or TGFb in the mucosal microenvironment (see⁹¹), similar to what has recently been shown for DC populations⁹². Furthermore mouse intestinal macrophages produce IL-10, both constitutively⁹³, and following stimulation with bacteria⁸⁷. In fact, IL-10 may be essential for this poor responsiveness of intestinal macrophages in mice since intestinal macrophages from IL-10−/− mice have enhanced cytokine production in response to TLR ligand stimulation^87,93,94. Therefore, intestinal macrophages in the steady-state are non-inflammatory cells that retain the capacity to phagocytose and kill invading microbes.

Recent data in the mouse suggest intestinal macrophages may also have the capacity to induce FoxP3+ Tregs⁹³. Thus, it was shown that intestinal macrophages (CD11c^loCD11b+F4/80+ cells) from the murine small intestine produced IL-10, and induced FoxP3+ Tregs from naïve T cells in culture, but only significantly in the presence of exogenously added TGFβ. Interestingly, in this study, Treg induction was IL-10 dependent, and DC populations, including CD11b− CD103+ DCs were not capable of inducing Tregs in the absence of TGFβ. Furthermore CD11b+ DCs, which are primarily CD103− induced Th17 cells, the induction of which was suppressed by the co-culture with intestinal macrophages. While it is not yet clear why this study provided discrepant results from studies from other laboratories with regard to CD103+ intestinal DC function^73,75, it indicates a potential additional suppressive function of intestinal macrophages. Local macrophages may be capable of supporting Treg and blocking Th17induction within the local tissues, while still allowing migrating CD103+ DCs to drive Treg induction once they reach the MLN.

During active intestinal inflammation, as occurs in Crohn’s disease in humans, blood monocytes are recruited to the inflamed tissues where they release a variety of proinflammatory cytokines, such as TNF-α, macrophage infiltrating factor (MIF), IL-1, IL-6, IL-12 and IL-18, which are critically involved in the onset and the development of Crohn's disease. Furthermore, during infectious inflammation of the intestine, such as that occurring with S. typhimurium infection in the mouse, a population of CD11cint CD11b+ cells is recruited to PPs that produce TNFα that are likely derived from monocytes⁹⁵, These cells may be similar to cell recruited to the spleen during listeria infection that produced TNFα and iNOS (so called “Tip DCs”)⁹⁶. Recently, these same cells have been implicated in the regulation of IgA production in mucosa-associated lymphoid tissues⁹⁷.

The definition and ontogeny of intestinal DC and macrophage populations

Comparatively little is know about the developmental and functional relationships between DC populations and macrophages in the intestine, in particular when compared to what is known about the spleen or skin.

This issue was recently addressed in cell transfer studies aimed at identify the progenitors of LP DCs. It is now clear that two subpopulations of blood monocytes exist in mice, humans, and rats, one that is CCR2+ CX3CR1^int, and another that is CCR2−CXCR3^hi (see⁹⁸). Both subsets in the mouse express F4/80 and CD11b. In addition, CCR2+ cells also express high levels of LyC6 (stained with GR1 antibody), and were originally shown to migrate to inflammatory sites (so-called “inflammatory” monocytes), while CCR2− cells express low levels of Ly6C migrate constitutively to non-inflammed sites, and both have the potential to give rise to DCs⁹⁹. In addition, the same group isolated a common progenitor from the BM that has the capacity to differentiate into both DCs and monocytes (MDP)¹⁰⁰,. Using adoptive transfer strategies with these defined cell poulations, Jung and colleagues demonstrated that CCR2+Ly6C^hi monocytes given IV to normal mice resulted in no detectable spleen or LP DCs derived from the transferred cells. However, when given to mice depleted of CD11c cells using the CD11c-diphtheria toxin receptor (DTR) transgenic mice given DT, LP DCs were readily replenished. In contrast, spleen DCs were only repopulated when given MDP and not CCR2+Ly6C^hi monocytes¹⁰¹ consistent with a self-renewing spleen DC progenitor described by Naik and colleagues¹⁰². Furthermore, the same was true for mice made genetically deficient form CD11c+ cells¹⁰¹. Furthermore all developing DCs were CX3CR1+ cells, similar to what has been shown for a large portion of LP DCs from normal mice, and in particular those that extend dendrites into the intestinal lumen²⁶.

These studies indicate that monocytes may be a source of LP and lung, but not spleen DCs under steady-state conditions. One obvious question remaining is whether monocytes can only give rise to LP DCs under conditions of depletion, as occurs for Langerhans’ cells in the skin following UV irradiation, since no LP DCs were detectable following adoptive transfer in normal wild-type mice. However, since the surface phenotype of the generated LP DCs was similar to what one finds in normal mice argues that this is not the case. In addition, since the CCR2+Ly6C^hi monocytes after transfer are short lived, return to the bone marrow and emerge as CCR2−Ly6C^lo cells, it is not clear which subpopulaton of monocyte is required for the repletion. These data suggest that conventional DCs in mucosal lymphoid tissues may be derived from different populations than LP DCs, and thus constitute fundamentally different cells. Interesting, the CD103+ DCs from the LP appear to overlap with those expressing CX3CR1, suggesting they may represent the monocyte derived cell population …under true steady-state conditions⁵⁵. Lastly, it was recently shown that some CD11c^hi as well as CD11c^int cell populations from the small intestinal LP express F4/80⁹². Whether these cell populations represent monocyte-derived cells is not at all clear, and raises caution in using the F4/80 as a marker of intestinal macrophages. Clearly more needs to be done to define the phenotype and ontological relationships of intestinal “DC” and “macrophage” populations.

CONCLUDING REMARKS

Recent studies point to a primary role for the local tissue environment in the conditioning of both DCs and macrophages in the steady-state to promote tissue specific immune responses that protect against pathology. In particular, factors produced by epithelial cells may be involved, as highlighted in several recent studies^103–106. These may include TSLP (IL-50), TGFβ, and others produced under continuous signaling induced by intestinal flora. In addition, IL-10 from macrophages^87,93 and DCs^69,92 acting in an autocrine or paracrine manner, as well as prostaglandin E₂ from stromal cells¹⁰⁷ may significantly influence DC and macrophage function. The end result of such conditioning is to affect DCs to drive less pathological Th2 and Treg responses, and to positively affect IgA production against commensal organisms, as well as for macrophages to act as innate cells by phagocytosing and killing bacteria.

How microenvironental conditioning of DCs and TLR signaling can be overcome to initiate positive immune responses to pathogens is not yet clear. However, the use of virulence factors by pathogenic bacteria¹⁰⁸ may induce the expression of chemokines, and inflammatory cytokines from epithelial¹⁰⁹ or other cells, likely resulting in the recruitment of innate immune cells including neutrophils and macrophages, as well new DC precursors¹¹⁰. Under these conditions, the change in the local milieu, including the production of IL-6, TNFα and IL1β would overcome regulatory effects of locally suppressive factors (TSLP, PGE₂, and TGFβ) to activate new DC migrants from the blood, and following their migration to MLNs or to T cell zones in mucosal follicles to drive effector T cells. Alternatively the local production of proinflammatory factors (including IL-6) may subvert effector T cell suppression by Tregs¹¹¹, or overcome the RA/TGFβ-dependent differentiation of naïve T cells into Tregs and drive them to effectors^74,77.

Clearly a better understanding of how to define DC and macrophage populations in the intestine, and how they function together within local inductive and effector tissues has the potential to contribute greatly to the development of new vaccines and treatments for intestinal inflammation.

REFERENCES