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The Multifaceted Personality of Intestinal CX3CR1+ Macrophages
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Opinion
The Multifaceted Personality of Intestinal CX3CR1+ Macrophages Mari Regoli,1 Eugenio Bertelli,1 Massimo Gulisano,2 and Claudio Nicoletti2,* Intestinal macrophages expressing the fraktalkine receptor (CX3CR1+[7_TD$IF]) represent a cell population that plays a variety of roles ranging from maintaining intestinal immune homeostasis at steady state to controlling antigen access by extending transepithelial dendrites (TEDs) to capture luminal microbes and shuttle them across the epithelium to initiate immune responses. However, recent evidence shows that very early during infection, pathogen-capturing CX3CR1+ macrophages migrate to the lumen of the small intestine, therefore preventing pathogens from traversing the epithelium. Here we discuss the complexity of the at-times seemingly opposing roles played by these cells and propose that CX3CR1-mediated pathogen exclusion is part of a defensive strategy against infections that includes multiple effector mechanisms acting synergistically at the intestinal mucosa.
Trends Intestinal macrophages expressing the fraktalkine receptor (CX3CR1+) play a variety of roles at the host–microbe interface in the gut. CX3CR1+ macrophages originate from Ly6Chi precursors and contribute to the maintenance of intestinal immune homeostasis. CX3CR1+ macrophages can sample antigen and migrate back to the lymph node, but also transmigrate to the lumen of the gut following pathogen uptake.
Protecting the Intestinal Mucosa and Sensing the Environment: Multiple Strategies at the Host–Microbe Interface Mammalian epithelial surfaces are colonized by large numbers of bacteria; however, it is the gastrointestinal (GI) tract that faces the most abundant bacterial burden as well as daily challenges by a large number and diversity of food antigens. The complexity of the environment has led to the evolution of strategies that include, on the one hand, mechanical barriers that prevent microbes and macromolecules from trespassing across the intestinal epithelia barrier and, on the other hand, the presence of portals through which the luminal contents can be continuously monitored to detect the presence of potentially harmful microbes [1]. Penetration of macromolecules and microbes into the intestinal tissue and consequently into the systemic circulation is prevented by the presence of several elements acting synergistically that form the intestinal epithelial barrier (Figure 1). First, the epithelium is formed by a single layer of cells joined by tight junctions that allow the passage of water and ions but provide an effective mechanical barrier to macromolecules and microbes [2]. Furthermore, as their name indicates, mucosal surfaces are covered by mucus locally produced by goblet cells. Mucus thickness increases greatly along the proximal–distal axis [3] and plays a critical role in preventing microbial interaction with/penetration of the epithelium [4,5]. Integral to the mucus layer is the abundant presence of secretory IgA (sIgA) antibody and antimicrobial peptides (AMPs) produced by intestinal epithelial cells (IECs) and Paneth cells [6,7]. Combined, these diverse effectors ensure that only a small percentage of microbes engage with the IECs at any time. However, appropriate mucosal and systemic immune responses can be mounted only on sampling of luminal contents. To this end a series of mechanisms for antigen sampling are present in the gut. Specialized antigen-sampling microfold (M) cells of the follicle-associated
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The intraluminal transmigration of CX3CR1+ macrophages significantly reduces the pathogen burden in the intestinal tissue, thus acting as a rapidly deployed mechanism of pathogen exclusion. CX3CR1+ macrophage-mediated antigen sampling and intraluminal migration occur in different areas of the gut and are triggered by different regulatory signals.
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Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy 2 Department of Experimental and Clinical Medicine, Section of Anatomy, University of Florence, Florence, Italy
*Correspondence: [email protected] (C. Nicoletti).
http://dx.doi.org/10.1016/j.it.2017.07.009 © 2017 Elsevier Ltd. All rights reserved.
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Figure 1. The Intestinal Epithelial Barrier: Between Exclusion and Sampling. The intestinal epithelial barrier comprises several components that are physical, biochemical, and immunological in nature. Commensals, the intestinal epithelium, and the underlying immune system work closely together to establish and maintain intestinal immune homeostasis. The intestinal epithelium is a single-cell layer that constitutes a selectively permeable barrier allowing the absorption of nutrients, ions, and water while blocking intraluminal toxins, antigens, and microbes. The epithelium is covered by a mucus layer rich in glycoproteins and its thickness varies between approximately 200 mm in the duodenum and 700–800 mm in the colon/rectum. Also, large amounts of soluble IgA (sIgA) are produced in the lamina propria (LP) and subsequently transported into the lumen by polyIg receptors. These components represent a formidable barrier to potentially harmful pathogens. However, to constantly survey the luminal contents, a series of strategies is also present. Pathogen recognition receptors (PRRs) such as Toll-like receptors (TLRs) on intestinal epithelial cells (IECs) and various strategies for antigen sampling (red boxes) located in different areas of the gastrointestinal (GI) tract allow the gut immune system to react promptly and generate rapid and effective immune responses in the presence of pathogenic organisms. Due to the large amount of antigenic material transported, the microfold (M) cells of the follicle-associated epithelium (FAE) are the most important antigen-sampling cells and deliver antigens directly to the lymphoid tissue where they can be handled by the immune system. Antigen sampling is also performed by CX3CR1+ macrophages inhabiting the lamina propria, a much smaller number villous M cells, and goblet cells. DC, dendritic cell.
epithelium (FAE) of Peyer’s patches (PPs) transport macromolecules and microorganisms to the underlying lymphoid tissue where the immune machinery is in place to tackle potentially harmful trespassers [8,9]. Antigen sampling is also performed by a smaller number of villusassociated M cells [10], goblet cells [11], and lamina propria (LP)-resident CX3CR1+ macrophages [12,13]. The latter cell type participates in antigen sampling by extending TEDs between epithelial cells to capture antigens or whole microbes and subsequently shuttle them across the epithelial barrier (Figure 2A). This event, also called antigen sampling via the indirect route, is facilitated by the expression of tight junction proteins by the sampling macrophages, which enables antigen sampling via TEDs without altering the integrity of the intestinal epithelium [12]. The ability of CX3CR1+ macrophages to sample luminal antigens, in addition to their role in the regulation of intestinal immune homeostasis and barrier function, has further highlighted the complexity of the wide array of biologically relevant functions of these cells in the intestinal mucosa.
The Busy Life of Intestinal CX3CR1+ Macrophages Intestinal CX3CR1+ macrophages have long been considered antigen-presenting dendritic cells (DCs) [13–15]; however, recent multiparameter flow cytometry studies and
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Figure 2. Dual Behavior of CX3CR1+ Macrophages at the Mucosal Interface in the Gut in Response to Pathogens. Intestinal CX3CR1+ macrophages respond to distinct signals originating from pathogens and tackle the offending microbes in different ways. In the presence of Salmonella Typhimurium, they can extend transepithelial dendrites between intestinal epithelial cells (IECs) and sample microbes to initiate antigen-specific immune responses (A). Alternatively, they can migrate into the intestinal lumen (B) and internalize microbes to prevent pathogens from traversing the epithelium and infecting the host. The exact series of events leading to either antigen sampling or migration mode is unclear; it seems that these events represent two distinct functions of the CX3CR1+ cells that are likely to intervene at different stages of infection in distinct areas of the gut. The microbial signals inducing antigen sampling include the engagement of Toll-like receptor 4 (TLR4) with its ligand LPS and do not include flagellin expression. Instead, transmigration of CX3CR1+ cells depends not on LPS but on the presence of flagellin and the ability of Salmonella to invade the host. The dynamics of the two events also differ. During antigen sampling the cell body remains located within the lamina propria (LP) beneath and in close contact with the epithelium and only a thin extracellular extension protrudes between the IECs. Instead, the process of CX3CR1+ cell transmigration involves the penetration and passage of whole cells between the IECs, suggesting the involvement of different adhesion molecules/integrins. However, CX3CR1+-mediated antigen sampling occurs in the terminal ileum and the colon, whereas transmigration occurs throughout the small intestine but is absent in the colon.
lineage-tracking strategies have established their nature as resident macrophages distinct from conventional aE integrin CD103+ DCs. LP CX3CR1+ macrophages represent a heterogeneous population that comprises a majority of cells expressing high levels of CX3CR1 (CX3CR1hi) alongside a minority, but still significant, population expressing intermediate levels of the receptor (CX3CR1int) [15,16]. These subsets represent different maturation stages of a cell population that originated from a common Ly6Chi precursor. At steady state CX3CR1int cells give rise to a short-lived anti-inflammatory CX3CR1hi cell population characterized by a noninflammatory gene expression profile that includes expression of IL-10, TREM-2, CD163,
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Stab1, MRC1 LYVE-1, TNFAIP2 (A20), and RETNLA (FIZZ-1). The differentiation pattern of these cells is highly influenced by the inflammatory status of the gut. During colitis the maturation of CX3CR1int into anti-inflammatory CX3CR1hi macrophages does not occur, leading to the accumulation of proinflammatory CX3CR1int cells in the LP [15]. These effector cells sense the environment and respond to bacterial stimuli via Toll-like receptors (TLRs) and NOD2 and promote inflammation [16]. Also, at a later stage they give rise to a phenotypically and functionally distinct CX3CR1int population that shows typical DC migratory properties including the acquisition and processing of antigens as well as the expression of CCR7, which enable them to travel to the mesenteric lymph nodes (MLN) to prime naïve CD4 T cells. Furthermore, experiments that utilized mice with CX3CR1 macrophage-restricted IL-10 or IL-10 receptor alpha (IL-10Ra) deficiency demonstrated that the ability of CX3CR1+[9_TD$IF] macrophages to control intestinal inflammation is linked to their ability to sense IL-10. The lack of IL-10R, but not of CX3CR1 macrophage-derived IL-10, prevented the conditioning of CX3CR1 macrophages leading to the spontaneous development of severe colitis [17]. Instead, the production of IL-10 by CX3CR1+ macrophages, along with their ability to promote the local (intestinal) expansion of FoxP3+ T regulatory (Treg) cells following their migration from the MLNs, appears to be critical for the establishment of tolerance to orally fed antigens [18]. However, it has been suggested that the establishment of tolerance to orally fed antigens also hinges on antigen transfer from CX3CR1+ macrophages to local conventional CD103+ DCs via a connexin-43 gap junction mechanism [19]. CX3CR1+ macrophages also contribute significantly to barrier integrity by integrating signals originating from the intestinal bacteria and delivering these signals to other LP-inhabiting cells. First, intestinal macrophages, in contrast to CD103+ DCs, appeared to be essential, at steady state, in supporting commensal-induced generation of CD4 T helper 17 (TH17) cells, a cell population that plays an important role in maintaining mucosal barriers and contributes to pathogen clearance at mucosal surfaces the loss of which has been linked to chronic inflammation and microbial translocation [20]. Second, it has been shown that, in response to microbial stimuli, CX3CR1+ macrophages produced greater amounts of IL-23 and IL-1b than conventional CD103+ DCs and in so doing support the production of IL-22 by group 3 innate lymphoid cells (ILC3s) [21,22], thus promoting mucosal healing and maintaining a fully functional intestinal barrier. Taken together these data show that CX3CR1+ macrophages are characterized by a high degree of adaptability to the ever-changing environment of the gut and are key players in the regulation of intestinal immune homeostasis and barrier integrity. In addition, the observation that CX3CR1hi macrophages also sample luminal antigens [12] suggested that these cells might also be critical for the initiation of antigen-specific immune responses. Currently, the dynamics of antigen sampling by CX3CR1hi and the ensuing events remains under debate. Initially, it was thought that sampling via the indirect route was followed by the migration of bacteria-loaded CX3CR1+ cells to the MLNs, where they presented antigens to T cells [21]. This hypothesis was later questioned by the observation that at steady state CX3CR1+ macrophages appear to be a resident population that does not migrate to the MLNs and possesses little capability to prime naïve T cells [23]. This led to the formulation of the hypothesis that, after capturing antigens via TEDs, CX3CR1+ macrophages pass them to neighboring LP CD103+ DCs expressing CCR7, which in turn journey to the MLNs [19]. However, more recently others have suggested that the migration of bacteria-carrying CX3CR1+ macrophages to the MLNs should not be excluded. Depletion of the microbiota by antibiotic treatment induced these cells to acquire migratory properties by expressing the chemokine receptor CCR7 and transport Salmonella Typhimurium (S. Typhimurium) to the
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MLNs [24]. Although controversy remains regarding the level of expression of CX3CR1 on bacteria-loaded cells travelling to the MLNs following the acquisition of S. Typhimurium [25], it was shown that these cells also express the costimulatory molecule CD80, suggesting a direct involvement in T cell priming and a broader role of sampling CX3CR1+ macrophages in the orchestration of immune responses [24]. However, the notion that microbiota-derived signals prevent bacteria-loaded CX3CR1+ macrophages from traveling to the MLN was further confirmed in a study on the increased translocation of commensals via goblet cell-associated passages (GAPs) in the colon following antibiotic treatment [26]. In this case microbes transported to the LP via GAPs were then transported to the MLNs almost exclusively by CX3CR1+CD103 macrophages. Remarkably, indirect sampling via TEDs is not the only event involving CX3CR1hi cells at the host–microbe interface in the gut. Under similar experimental circumstances (i.e., challenge with non-pathogenic InvA Salmonella), intraluminal transmigration of resident Salmonellacapturing CX3CR1hi cells occurred in the small intestine very early post-infection (Figure 2B) [27,28]. This luminal transmigration led to a significant reduction in the number of pathogens traversing the epithelial barrier [29], suggesting an important role in the protection of the intestinal mucosa.
To Move Back In or Keep Traveling Out: That Is the Dilemma The notion that the same antigenic stimulus triggers two distinct and seemingly opposing behaviors of CX3CR1+ macrophages in the small intestine shows the complexity of the signaling network operating at the mucosal host–microbe interface. It also raises a series of questions, the most compelling being: what regulates the decision [3_TD$IF]of a CX3CR1+[10_TD$IF] cell to move out into the lumen to capture pathogens or to protrude temporarily into the lumen and then retract the TED to initiate immune responses? A definitive answer to this issue remains elusive; however, it appears that the two events are under the control of different mechanisms, suggesting that they might be distinct functions of CX3CR1+ macrophages. First, the formation of TEDs is promoted by the engagement of soluble flagellin with TLR4 [12,30]; it was found instead that non-pathogenic Escherichia coli and its associated LPS did not induce CX3CR1+ macrophage trafficking across the epithelium [27]. Second, it was shown that the formation of TEDs is independent of the engagement of flagellin with its receptor, TLR5 [30]. By contrast, it was determined that Salmonella-bound flagellin played a critical role in CX3CR1+ transepithelial migration, because a Salmonella mutant that did not express the FliC and FljB flagellins failed to trigger intraluminal transmigration [27]. These observations show that LPS and flagellin play different roles in the formation of TEDs and transmigration of CX3CR1+ cells. Also, taken together these findings suggest that multiple signals from the offending pathogen are required to trigger the intraluminal transmigration of CX3CR1+ macrophages [27]. Further, the role of flagellin was confirmed by a series of experiments that showed the absence of macrophage transmigration on challenge with an SPI1- and SPI2-deficient Salmonella (DSPI1DssrA) double mutant and in mice lacking the adaptor molecule MyD88 solely in IECs (MyD88DIEC) [29]. Importantly, the non-flagellated Salmonella mutant retained the ability to translocate across the epithelial barrier. The latter notion raised the possibility that cells other than CX3CR1+ macrophages participate in indirect antigen sampling. Observations from several investigators lent support to this hypothesis. Chieppa et al. observed [30], in contrast to the report of Niess et al. [13], TEDs in Salmonella-treated CX3CR1gfp/gfp mice [31]. However, canonical CX3CR1 CD103+ DCs have also been observed to extend TEDs through the intestinal epithelium and acquire luminal Salmonella [32]. The possibility that other cells inhabiting the LP are capable of producing TEDs could also explain the observation that the lack of CX3CR1-dependent TEDs affected neither pathogen entry nor subsequent T cell priming in the MLNs [23,32]. Remarkably, in contrast to CX3CR1
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transmigration, the ability to form TEDs depends on the genetic make-up of the host [33], an observation that led to the conclusion that CX3CR1-mediated sampling might not be a universal phenomenon [34]. Furthermore, indirect sampling appeared to be present in the terminal ileum [13] and colon [35] whereas CX3CR1 migration occurred throughout the small intestine but was absent or negligible in the colon [27,28]. Taken together these observations imply that CX3CR1-mediated sampling and transmigration are independent events regulated by as-yet-unidentified signals that might intervene at different stages of the infection in geographically distinct areas of the gut.
Blocking Unwanted Visitors: Where Do Intraluminal CX3CR1+ Macrophages Fit? Reduction of intestinal bacterial load showed that the transmigration of CX3CR1+ cells is a rapidly deployed cell-based mechanism of pathogen exclusion. This defensive response is only one of the strategies implemented at the host–microbe interface in the gut for the prevention of infection by pathogens (Figure 3). Pathogenic microbes trigger rapid host production of AMPs [36,37], mucus secretion [38], and recruitment of innate immune cells such as neutrophils [39]. However, a wide variety of microbes have evolved strategies, including high motility and a type-III section system (TTSS-1) [40], to bypass the preventive mechanical/biochemical defenses and breach the intestinal epithelial barrier. The danger signals emerging from the latter event trigger further action to limit pathogen invasion. Very rapidly (15–30 min) post-infection, a fairly large contingent of CX3CR1+ macrophages migrates into the intestinal lumen, significantly reducing the Salmonella load in the intestinal tissue [27,28]. Evidence indicated the migration of CX3CR1+ cells as a protective emergency response occurring once the mucus–epithelium barrier has been overcome by offending microbes; the migration of CX3CR1+ was more pronounced in response to wild-type invasive Salmonella that the non-invasive InvA strain [29]. Furthermore, in migration-deficient CX3CR1 / mice the lack of intraluminal CX3CR1+ cells allowed a higher proportion of Salmonella to trespass the epithelium. This is likely to explain their greater susceptibility to Salmonella infection compared with their wild-type counterparts [13,29]. Conventional microscopy techniques and in vivo real-time imaging showed that several CX3CR1+ macrophages transmigrate in single file through the same paracellular channel into the lumen [29]. This observation makes it unlikely, at least at this stage, that the CX3CR1+ macrophage can retract the sampling TED and either move back to the MLN or transfer the captured antigen to bystander conventional CD103+ DCs. In this scenario one also has to consider the possibility that the intraluminal transmigration is merely a physical consequence of the mechanical pressure exerted by CX3CR1+ cells migrating towards the intestinal lumen to perform antigen sampling. However, it seems unlikely that this transmigration represents an accidental event as it would inevitably lead to the loss of cells that are instrumental in regulating immune intestinal homeostasis and barrier integrity. Although the exact mechanisms utilized by intraluminal CX3CR1+ cells to reduce the number of pathogens in the intestinal tissue remains to be determined, it is likely that this is achieved either by killing the internalized pathogens or by transporting them to the colon where the much thicker mucus layer affords better protection and where the abundant gut microbiota can reduce pathogen viability [41]. In this context the observation that CX3CR1+ transmigration is restricted to the small intestine is of interest. Although the mechanisms underpinning the differential behavior of CX3CR1+ macrophages in response to pathogen challenge in different areas of the gut remain to be determined, it is possible that signals emerging from IEC– Salmonella engagement in the colon differ intrinsically from those in the small intestine.
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Figure 3. Multilayered Strategy of Pathogen Exclusion in the Gut. Preventing the penetration of potentially harmful microbes is one of the most important functions of the intestinal epithelial barrier. The exclusion of pathogens at the mucosal interface in the gut can be divided into three major steps: preventive, rapid, and delayed responses. At steady state intestinal epithelial cells (IECs) and the goblet cell-derived mucus layer provide an efficient preventive (A) biochemical/mechanical barrier to potentially harmful microbes. The mucus is enriched by the abundant production of secretory IgA (sIgA) antibody secreted continuously by plasma cells inhabiting the lamina propria and by antimicrobial peptides (AMPs), which are critical to the regulation of microbial access to the gut epithelium (B). A rapid response occurs within minutes of pathogens breaching the epithelial barrier and initiating mucosal infection (C). Very soon after infection (15–30 min), CX3CR1+ macrophages transmigrate into the intestinal lumen and internalize the luminal pathogens. This rapidly deployed cell-based mechanism of pathogen exclusion significantly limits the number of pathogens infecting the host and reaches its peak 5 h after infection. Preventive barriers and the rapid response are followed, if necessary, by a delayed response (D). Pathogens that have escaped the initial host defenses and managed to infect the epithelial cells trigger (starting 8 h post-infection) epithelium-intrinsic NAIP/NLRC4 inflammasome-driven expulsion of infected cells (D). Both the rapid and the delayed response have been observed in response to Salmonella infection; it remains to be determined whether these are Salmonella-specific responses or a general protective mechanism against pathogens. pIgR, polyIg receptor.
Alternatively, the abundant colonic mucus layer and microbiota might prevent or reduce the recruitment of transmigrating CX3CR1+ macrophages. Ultimately however, this implies that CX3CR1-mediated pathogen exclusion occurs in a microbial environment far less complex than the colon where pathogens would not be easily accessible to pathogen-capturing CX3CR1+ macrophages. The latter cell type is the first intraluminal population found to colocalize with Salmonella [27]; at a later stage, other cell types, such as neutrophils, also transmigrate into the intestinal lumen [39]. However, important differences between intraluminal CX3CR1+ cells and neutrophils exist. Intraluminal neutrophils do not eliminate Salmonella in the gut [42]; further, in Toxoplasma gondii infection infected neutrophils that have migrated into the intestinal lumen can transmigrate back into the tissue, contributing to parasite spread [43]. By contrast, CX3CR1+ cells embark on a unidirectional migration and do not cross back into the intestinal tissue [29]. To prevent pathogens from traversing the epithelial barrier is a key goal and the rapid protective response mediated by CX3CR1+ macrophages is followed by an additional defensive strategy that restricts the replicative niche established by Salmonella that have managed to infect the intestinal mucosa. Pathogen-mediated CX3CR1 transmigration reached its peak after 5 h and
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declined at 12 h post-infection [29]. At a later time point, Sellin et al. [44] showed that, starting from 8 h post-infection, epithelial EpCAM+ cells harboring Salmonella could be observed in different stages of dislodging from the mucosa and are eventually expelled into the intestinal lumen. Mechanistically, the expulsion of infected IECs is driven by an epithelium-intrinsic defense axis dependent on the NAIP/NLRC4 inflammasome. Further quantitative and timecourse analyses clearly demonstrated the contribution of this mechanism to the reduction of intraepithelial Salmonella load. Also, although the expulsion mechanism closely mimics the physiological process of IEC shedding on cell death, a much larger percentage of expelled IECs contained intraluminal Salmonella, demonstrating that the expulsion preferentially targeted infected IECs. Like CX3CR1 transmigration, this inflammasome-triggered expulsion is dependent on flagellin, a NAIP ligand, and on Salmonella’s ability to invade.
Concluding Remarks Intestinal CX3CR1+ macrophages are a multitasking cell population that plays a central role in controlling intestinal immune homeostasis and barrier function and participates in mucosal defense and clearance. On infection with S. Typhimurium, these cells can discriminate between the different signals produced by the pathogens and, depending on the stage of infection and most likely on the geographical location in the gut, they can either extend TEDs to sample luminal antigens or migrate into the intestinal lumen to prevent Salmonella from traversing the epithelium. Dissecting in detail the underpinning regulatory signaling network will help us ascertain the respective roles of these two CX3CR1-mediated events in combating infections (see Outstanding Question). At this stage it is plausible to hypothesize that the transmigration of Salmonella-capturing CX3CR1+ macrophages is a rapid response that intervenes once the integrity of the epithelial barrier has been breached and that is part of a multilayered defensive strategy including preventive mechanical/biochemical barriers followed, if necessary, by additional measures (i.e., expulsion of infected IECs) to reduce the pathogen burden in intestinal tissue. Acknowledgments The authors thank P. Pople and U. Santosuosso for their help with computer artwork.
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Outstanding Questions What are the distinct microbial signals triggering antigen sampling or intraluminal migration by the same cell type? Also, given the different behaviors of CX3CR1+ macrophages in response to the same antigenic stimuli in geographically distinct areas of the gut, is there a local control that triggers these seemingly opposing functions? Finally, is the lack of Salmonellainduced transmigration in the colon due to an intrinsically different response of colonic IECs to Salmonella infection? Both indirect CX3CR1+ macrophagemediated sampling and migration have been studied so far in response to Salmonella. Are these Salmonella-specific events or do they represent a more general protective response to pathogens? During antigen sampling the body of CX3CR1+ macrophages remains in the LP and only the TED protrudes between IECs, whereas during migration the whole cell body migrates between IECs. Are different adhesion molecules/integrins involved in the dynamics of the two events? Is CX3CR1-mediated antigen sampling indispensable for mounting an antigen-specific response? Do intraluminal CX3CR1+ cells eliminate/neutralize pathogens by direct killing or by transporting them to areas of the gut characterized by a thicker mucus layer and more abundant microbiota that can have a deleterious effect on pathogen viability?
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39. Fournier, B.M. and Parkos, C.A. (2012) The role of neutrophils during intestinal inflammation. Mucosal Immunol. 5, 354–366 40. Galán, J.E. and Wolf-Watz, H. (2006) Protein delivery into eukaryotic cells by type III secretion machines. Nature 444, 567–573 41. Avendaño-Pérez, G. et al. (2015) Interactions of Salmonella enterica subspecies enterica serovar Typhimurium with gut bacteria. Anaerobe 33, 90–97 42. Loetscher, Y. et al. (2012) Salmonella transiently reside in luminal neutrophils in the inflamed gut. PLoS One 7, e34812 43. Coombes, J.L. et al. (2013) Motile invaded neutrophils in the small intestine of Toxoplasma gondii-infected mice reveal a potential mechanism for parasite spread. Proc. Natl. Acad. Sci. U. S. A. 110, E1913–E1922 44. Sellin, M.E. et al. (2014) Epithelium-intrinsic NAIP/NLRC4 inflammasome drives infected enterocyte expulsion to restrict Salmonella replication in the intestinal mucosa. Cell Host Microbe 16, 237–248
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The Multifaceted Personality of Intestinal CX3CR1+ Macrophages Mari Regoli,1 Eugenio Bertelli,1 Massimo Gulisano,2 and Claudio Nicoletti2,* Intestinal macrophages expressing the fraktalkine receptor (CX3CR1+[7_TD$IF]) represent a cell population that plays a variety of roles ranging from maintaining intestinal immune homeostasis at steady state to controlling antigen access by extending transepithelial dendrites (TEDs) to capture luminal microbes and shuttle them across the epithelium to initiate immune responses. However, recent evidence shows that very early during infection, pathogen-capturing CX3CR1+ macrophages migrate to the lumen of the small intestine, therefore preventing pathogens from traversing the epithelium. Here we discuss the complexity of the at-times seemingly opposing roles played by these cells and propose that CX3CR1-mediated pathogen exclusion is part of a defensive strategy against infections that includes multiple effector mechanisms acting synergistically at the intestinal mucosa.
Trends Intestinal macrophages expressing the fraktalkine receptor (CX3CR1+) play a variety of roles at the host–microbe interface in the gut. CX3CR1+ macrophages originate from Ly6Chi precursors and contribute to the maintenance of intestinal immune homeostasis. CX3CR1+ macrophages can sample antigen and migrate back to the lymph node, but also transmigrate to the lumen of the gut following pathogen uptake.
Protecting the Intestinal Mucosa and Sensing the Environment: Multiple Strategies at the Host–Microbe Interface Mammalian epithelial surfaces are colonized by large numbers of bacteria; however, it is the gastrointestinal (GI) tract that faces the most abundant bacterial burden as well as daily challenges by a large number and diversity of food antigens. The complexity of the environment has led to the evolution of strategies that include, on the one hand, mechanical barriers that prevent microbes and macromolecules from trespassing across the intestinal epithelia barrier and, on the other hand, the presence of portals through which the luminal contents can be continuously monitored to detect the presence of potentially harmful microbes [1]. Penetration of macromolecules and microbes into the intestinal tissue and consequently into the systemic circulation is prevented by the presence of several elements acting synergistically that form the intestinal epithelial barrier (Figure 1). First, the epithelium is formed by a single layer of cells joined by tight junctions that allow the passage of water and ions but provide an effective mechanical barrier to macromolecules and microbes [2]. Furthermore, as their name indicates, mucosal surfaces are covered by mucus locally produced by goblet cells. Mucus thickness increases greatly along the proximal–distal axis [3] and plays a critical role in preventing microbial interaction with/penetration of the epithelium [4,5]. Integral to the mucus layer is the abundant presence of secretory IgA (sIgA) antibody and antimicrobial peptides (AMPs) produced by intestinal epithelial cells (IECs) and Paneth cells [6,7]. Combined, these diverse effectors ensure that only a small percentage of microbes engage with the IECs at any time. However, appropriate mucosal and systemic immune responses can be mounted only on sampling of luminal contents. To this end a series of mechanisms for antigen sampling are present in the gut. Specialized antigen-sampling microfold (M) cells of the follicle-associated
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The intraluminal transmigration of CX3CR1+ macrophages significantly reduces the pathogen burden in the intestinal tissue, thus acting as a rapidly deployed mechanism of pathogen exclusion. CX3CR1+ macrophage-mediated antigen sampling and intraluminal migration occur in different areas of the gut and are triggered by different regulatory signals.
1
Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy 2 Department of Experimental and Clinical Medicine, Section of Anatomy, University of Florence, Florence, Italy
*Correspondence: [email protected] (C. Nicoletti).
http://dx.doi.org/10.1016/j.it.2017.07.009 © 2017 Elsevier Ltd. All rights reserved.
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Figure 1. The Intestinal Epithelial Barrier: Between Exclusion and Sampling. The intestinal epithelial barrier comprises several components that are physical, biochemical, and immunological in nature. Commensals, the intestinal epithelium, and the underlying immune system work closely together to establish and maintain intestinal immune homeostasis. The intestinal epithelium is a single-cell layer that constitutes a selectively permeable barrier allowing the absorption of nutrients, ions, and water while blocking intraluminal toxins, antigens, and microbes. The epithelium is covered by a mucus layer rich in glycoproteins and its thickness varies between approximately 200 mm in the duodenum and 700–800 mm in the colon/rectum. Also, large amounts of soluble IgA (sIgA) are produced in the lamina propria (LP) and subsequently transported into the lumen by polyIg receptors. These components represent a formidable barrier to potentially harmful pathogens. However, to constantly survey the luminal contents, a series of strategies is also present. Pathogen recognition receptors (PRRs) such as Toll-like receptors (TLRs) on intestinal epithelial cells (IECs) and various strategies for antigen sampling (red boxes) located in different areas of the gastrointestinal (GI) tract allow the gut immune system to react promptly and generate rapid and effective immune responses in the presence of pathogenic organisms. Due to the large amount of antigenic material transported, the microfold (M) cells of the follicle-associated epithelium (FAE) are the most important antigen-sampling cells and deliver antigens directly to the lymphoid tissue where they can be handled by the immune system. Antigen sampling is also performed by CX3CR1+ macrophages inhabiting the lamina propria, a much smaller number villous M cells, and goblet cells. DC, dendritic cell.
epithelium (FAE) of Peyer’s patches (PPs) transport macromolecules and microorganisms to the underlying lymphoid tissue where the immune machinery is in place to tackle potentially harmful trespassers [8,9]. Antigen sampling is also performed by a smaller number of villusassociated M cells [10], goblet cells [11], and lamina propria (LP)-resident CX3CR1+ macrophages [12,13]. The latter cell type participates in antigen sampling by extending TEDs between epithelial cells to capture antigens or whole microbes and subsequently shuttle them across the epithelial barrier (Figure 2A). This event, also called antigen sampling via the indirect route, is facilitated by the expression of tight junction proteins by the sampling macrophages, which enables antigen sampling via TEDs without altering the integrity of the intestinal epithelium [12]. The ability of CX3CR1+ macrophages to sample luminal antigens, in addition to their role in the regulation of intestinal immune homeostasis and barrier function, has further highlighted the complexity of the wide array of biologically relevant functions of these cells in the intestinal mucosa.
The Busy Life of Intestinal CX3CR1+ Macrophages Intestinal CX3CR1+ macrophages have long been considered antigen-presenting dendritic cells (DCs) [13–15]; however, recent multiparameter flow cytometry studies and
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Figure 2. Dual Behavior of CX3CR1+ Macrophages at the Mucosal Interface in the Gut in Response to Pathogens. Intestinal CX3CR1+ macrophages respond to distinct signals originating from pathogens and tackle the offending microbes in different ways. In the presence of Salmonella Typhimurium, they can extend transepithelial dendrites between intestinal epithelial cells (IECs) and sample microbes to initiate antigen-specific immune responses (A). Alternatively, they can migrate into the intestinal lumen (B) and internalize microbes to prevent pathogens from traversing the epithelium and infecting the host. The exact series of events leading to either antigen sampling or migration mode is unclear; it seems that these events represent two distinct functions of the CX3CR1+ cells that are likely to intervene at different stages of infection in distinct areas of the gut. The microbial signals inducing antigen sampling include the engagement of Toll-like receptor 4 (TLR4) with its ligand LPS and do not include flagellin expression. Instead, transmigration of CX3CR1+ cells depends not on LPS but on the presence of flagellin and the ability of Salmonella to invade the host. The dynamics of the two events also differ. During antigen sampling the cell body remains located within the lamina propria (LP) beneath and in close contact with the epithelium and only a thin extracellular extension protrudes between the IECs. Instead, the process of CX3CR1+ cell transmigration involves the penetration and passage of whole cells between the IECs, suggesting the involvement of different adhesion molecules/integrins. However, CX3CR1+-mediated antigen sampling occurs in the terminal ileum and the colon, whereas transmigration occurs throughout the small intestine but is absent in the colon.
lineage-tracking strategies have established their nature as resident macrophages distinct from conventional aE integrin CD103+ DCs. LP CX3CR1+ macrophages represent a heterogeneous population that comprises a majority of cells expressing high levels of CX3CR1 (CX3CR1hi) alongside a minority, but still significant, population expressing intermediate levels of the receptor (CX3CR1int) [15,16]. These subsets represent different maturation stages of a cell population that originated from a common Ly6Chi precursor. At steady state CX3CR1int cells give rise to a short-lived anti-inflammatory CX3CR1hi cell population characterized by a noninflammatory gene expression profile that includes expression of IL-10, TREM-2, CD163,
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Stab1, MRC1 LYVE-1, TNFAIP2 (A20), and RETNLA (FIZZ-1). The differentiation pattern of these cells is highly influenced by the inflammatory status of the gut. During colitis the maturation of CX3CR1int into anti-inflammatory CX3CR1hi macrophages does not occur, leading to the accumulation of proinflammatory CX3CR1int cells in the LP [15]. These effector cells sense the environment and respond to bacterial stimuli via Toll-like receptors (TLRs) and NOD2 and promote inflammation [16]. Also, at a later stage they give rise to a phenotypically and functionally distinct CX3CR1int population that shows typical DC migratory properties including the acquisition and processing of antigens as well as the expression of CCR7, which enable them to travel to the mesenteric lymph nodes (MLN) to prime naïve CD4 T cells. Furthermore, experiments that utilized mice with CX3CR1 macrophage-restricted IL-10 or IL-10 receptor alpha (IL-10Ra) deficiency demonstrated that the ability of CX3CR1+[9_TD$IF] macrophages to control intestinal inflammation is linked to their ability to sense IL-10. The lack of IL-10R, but not of CX3CR1 macrophage-derived IL-10, prevented the conditioning of CX3CR1 macrophages leading to the spontaneous development of severe colitis [17]. Instead, the production of IL-10 by CX3CR1+ macrophages, along with their ability to promote the local (intestinal) expansion of FoxP3+ T regulatory (Treg) cells following their migration from the MLNs, appears to be critical for the establishment of tolerance to orally fed antigens [18]. However, it has been suggested that the establishment of tolerance to orally fed antigens also hinges on antigen transfer from CX3CR1+ macrophages to local conventional CD103+ DCs via a connexin-43 gap junction mechanism [19]. CX3CR1+ macrophages also contribute significantly to barrier integrity by integrating signals originating from the intestinal bacteria and delivering these signals to other LP-inhabiting cells. First, intestinal macrophages, in contrast to CD103+ DCs, appeared to be essential, at steady state, in supporting commensal-induced generation of CD4 T helper 17 (TH17) cells, a cell population that plays an important role in maintaining mucosal barriers and contributes to pathogen clearance at mucosal surfaces the loss of which has been linked to chronic inflammation and microbial translocation [20]. Second, it has been shown that, in response to microbial stimuli, CX3CR1+ macrophages produced greater amounts of IL-23 and IL-1b than conventional CD103+ DCs and in so doing support the production of IL-22 by group 3 innate lymphoid cells (ILC3s) [21,22], thus promoting mucosal healing and maintaining a fully functional intestinal barrier. Taken together these data show that CX3CR1+ macrophages are characterized by a high degree of adaptability to the ever-changing environment of the gut and are key players in the regulation of intestinal immune homeostasis and barrier integrity. In addition, the observation that CX3CR1hi macrophages also sample luminal antigens [12] suggested that these cells might also be critical for the initiation of antigen-specific immune responses. Currently, the dynamics of antigen sampling by CX3CR1hi and the ensuing events remains under debate. Initially, it was thought that sampling via the indirect route was followed by the migration of bacteria-loaded CX3CR1+ cells to the MLNs, where they presented antigens to T cells [21]. This hypothesis was later questioned by the observation that at steady state CX3CR1+ macrophages appear to be a resident population that does not migrate to the MLNs and possesses little capability to prime naïve T cells [23]. This led to the formulation of the hypothesis that, after capturing antigens via TEDs, CX3CR1+ macrophages pass them to neighboring LP CD103+ DCs expressing CCR7, which in turn journey to the MLNs [19]. However, more recently others have suggested that the migration of bacteria-carrying CX3CR1+ macrophages to the MLNs should not be excluded. Depletion of the microbiota by antibiotic treatment induced these cells to acquire migratory properties by expressing the chemokine receptor CCR7 and transport Salmonella Typhimurium (S. Typhimurium) to the
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MLNs [24]. Although controversy remains regarding the level of expression of CX3CR1 on bacteria-loaded cells travelling to the MLNs following the acquisition of S. Typhimurium [25], it was shown that these cells also express the costimulatory molecule CD80, suggesting a direct involvement in T cell priming and a broader role of sampling CX3CR1+ macrophages in the orchestration of immune responses [24]. However, the notion that microbiota-derived signals prevent bacteria-loaded CX3CR1+ macrophages from traveling to the MLN was further confirmed in a study on the increased translocation of commensals via goblet cell-associated passages (GAPs) in the colon following antibiotic treatment [26]. In this case microbes transported to the LP via GAPs were then transported to the MLNs almost exclusively by CX3CR1+CD103 macrophages. Remarkably, indirect sampling via TEDs is not the only event involving CX3CR1hi cells at the host–microbe interface in the gut. Under similar experimental circumstances (i.e., challenge with non-pathogenic InvA Salmonella), intraluminal transmigration of resident Salmonellacapturing CX3CR1hi cells occurred in the small intestine very early post-infection (Figure 2B) [27,28]. This luminal transmigration led to a significant reduction in the number of pathogens traversing the epithelial barrier [29], suggesting an important role in the protection of the intestinal mucosa.
To Move Back In or Keep Traveling Out: That Is the Dilemma The notion that the same antigenic stimulus triggers two distinct and seemingly opposing behaviors of CX3CR1+ macrophages in the small intestine shows the complexity of the signaling network operating at the mucosal host–microbe interface. It also raises a series of questions, the most compelling being: what regulates the decision [3_TD$IF]of a CX3CR1+[10_TD$IF] cell to move out into the lumen to capture pathogens or to protrude temporarily into the lumen and then retract the TED to initiate immune responses? A definitive answer to this issue remains elusive; however, it appears that the two events are under the control of different mechanisms, suggesting that they might be distinct functions of CX3CR1+ macrophages. First, the formation of TEDs is promoted by the engagement of soluble flagellin with TLR4 [12,30]; it was found instead that non-pathogenic Escherichia coli and its associated LPS did not induce CX3CR1+ macrophage trafficking across the epithelium [27]. Second, it was shown that the formation of TEDs is independent of the engagement of flagellin with its receptor, TLR5 [30]. By contrast, it was determined that Salmonella-bound flagellin played a critical role in CX3CR1+ transepithelial migration, because a Salmonella mutant that did not express the FliC and FljB flagellins failed to trigger intraluminal transmigration [27]. These observations show that LPS and flagellin play different roles in the formation of TEDs and transmigration of CX3CR1+ cells. Also, taken together these findings suggest that multiple signals from the offending pathogen are required to trigger the intraluminal transmigration of CX3CR1+ macrophages [27]. Further, the role of flagellin was confirmed by a series of experiments that showed the absence of macrophage transmigration on challenge with an SPI1- and SPI2-deficient Salmonella (DSPI1DssrA) double mutant and in mice lacking the adaptor molecule MyD88 solely in IECs (MyD88DIEC) [29]. Importantly, the non-flagellated Salmonella mutant retained the ability to translocate across the epithelial barrier. The latter notion raised the possibility that cells other than CX3CR1+ macrophages participate in indirect antigen sampling. Observations from several investigators lent support to this hypothesis. Chieppa et al. observed [30], in contrast to the report of Niess et al. [13], TEDs in Salmonella-treated CX3CR1gfp/gfp mice [31]. However, canonical CX3CR1 CD103+ DCs have also been observed to extend TEDs through the intestinal epithelium and acquire luminal Salmonella [32]. The possibility that other cells inhabiting the LP are capable of producing TEDs could also explain the observation that the lack of CX3CR1-dependent TEDs affected neither pathogen entry nor subsequent T cell priming in the MLNs [23,32]. Remarkably, in contrast to CX3CR1
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transmigration, the ability to form TEDs depends on the genetic make-up of the host [33], an observation that led to the conclusion that CX3CR1-mediated sampling might not be a universal phenomenon [34]. Furthermore, indirect sampling appeared to be present in the terminal ileum [13] and colon [35] whereas CX3CR1 migration occurred throughout the small intestine but was absent or negligible in the colon [27,28]. Taken together these observations imply that CX3CR1-mediated sampling and transmigration are independent events regulated by as-yet-unidentified signals that might intervene at different stages of the infection in geographically distinct areas of the gut.
Blocking Unwanted Visitors: Where Do Intraluminal CX3CR1+ Macrophages Fit? Reduction of intestinal bacterial load showed that the transmigration of CX3CR1+ cells is a rapidly deployed cell-based mechanism of pathogen exclusion. This defensive response is only one of the strategies implemented at the host–microbe interface in the gut for the prevention of infection by pathogens (Figure 3). Pathogenic microbes trigger rapid host production of AMPs [36,37], mucus secretion [38], and recruitment of innate immune cells such as neutrophils [39]. However, a wide variety of microbes have evolved strategies, including high motility and a type-III section system (TTSS-1) [40], to bypass the preventive mechanical/biochemical defenses and breach the intestinal epithelial barrier. The danger signals emerging from the latter event trigger further action to limit pathogen invasion. Very rapidly (15–30 min) post-infection, a fairly large contingent of CX3CR1+ macrophages migrates into the intestinal lumen, significantly reducing the Salmonella load in the intestinal tissue [27,28]. Evidence indicated the migration of CX3CR1+ cells as a protective emergency response occurring once the mucus–epithelium barrier has been overcome by offending microbes; the migration of CX3CR1+ was more pronounced in response to wild-type invasive Salmonella that the non-invasive InvA strain [29]. Furthermore, in migration-deficient CX3CR1 / mice the lack of intraluminal CX3CR1+ cells allowed a higher proportion of Salmonella to trespass the epithelium. This is likely to explain their greater susceptibility to Salmonella infection compared with their wild-type counterparts [13,29]. Conventional microscopy techniques and in vivo real-time imaging showed that several CX3CR1+ macrophages transmigrate in single file through the same paracellular channel into the lumen [29]. This observation makes it unlikely, at least at this stage, that the CX3CR1+ macrophage can retract the sampling TED and either move back to the MLN or transfer the captured antigen to bystander conventional CD103+ DCs. In this scenario one also has to consider the possibility that the intraluminal transmigration is merely a physical consequence of the mechanical pressure exerted by CX3CR1+ cells migrating towards the intestinal lumen to perform antigen sampling. However, it seems unlikely that this transmigration represents an accidental event as it would inevitably lead to the loss of cells that are instrumental in regulating immune intestinal homeostasis and barrier integrity. Although the exact mechanisms utilized by intraluminal CX3CR1+ cells to reduce the number of pathogens in the intestinal tissue remains to be determined, it is likely that this is achieved either by killing the internalized pathogens or by transporting them to the colon where the much thicker mucus layer affords better protection and where the abundant gut microbiota can reduce pathogen viability [41]. In this context the observation that CX3CR1+ transmigration is restricted to the small intestine is of interest. Although the mechanisms underpinning the differential behavior of CX3CR1+ macrophages in response to pathogen challenge in different areas of the gut remain to be determined, it is possible that signals emerging from IEC– Salmonella engagement in the colon differ intrinsically from those in the small intestine.
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Figure 3. Multilayered Strategy of Pathogen Exclusion in the Gut. Preventing the penetration of potentially harmful microbes is one of the most important functions of the intestinal epithelial barrier. The exclusion of pathogens at the mucosal interface in the gut can be divided into three major steps: preventive, rapid, and delayed responses. At steady state intestinal epithelial cells (IECs) and the goblet cell-derived mucus layer provide an efficient preventive (A) biochemical/mechanical barrier to potentially harmful microbes. The mucus is enriched by the abundant production of secretory IgA (sIgA) antibody secreted continuously by plasma cells inhabiting the lamina propria and by antimicrobial peptides (AMPs), which are critical to the regulation of microbial access to the gut epithelium (B). A rapid response occurs within minutes of pathogens breaching the epithelial barrier and initiating mucosal infection (C). Very soon after infection (15–30 min), CX3CR1+ macrophages transmigrate into the intestinal lumen and internalize the luminal pathogens. This rapidly deployed cell-based mechanism of pathogen exclusion significantly limits the number of pathogens infecting the host and reaches its peak 5 h after infection. Preventive barriers and the rapid response are followed, if necessary, by a delayed response (D). Pathogens that have escaped the initial host defenses and managed to infect the epithelial cells trigger (starting 8 h post-infection) epithelium-intrinsic NAIP/NLRC4 inflammasome-driven expulsion of infected cells (D). Both the rapid and the delayed response have been observed in response to Salmonella infection; it remains to be determined whether these are Salmonella-specific responses or a general protective mechanism against pathogens. pIgR, polyIg receptor.
Alternatively, the abundant colonic mucus layer and microbiota might prevent or reduce the recruitment of transmigrating CX3CR1+ macrophages. Ultimately however, this implies that CX3CR1-mediated pathogen exclusion occurs in a microbial environment far less complex than the colon where pathogens would not be easily accessible to pathogen-capturing CX3CR1+ macrophages. The latter cell type is the first intraluminal population found to colocalize with Salmonella [27]; at a later stage, other cell types, such as neutrophils, also transmigrate into the intestinal lumen [39]. However, important differences between intraluminal CX3CR1+ cells and neutrophils exist. Intraluminal neutrophils do not eliminate Salmonella in the gut [42]; further, in Toxoplasma gondii infection infected neutrophils that have migrated into the intestinal lumen can transmigrate back into the tissue, contributing to parasite spread [43]. By contrast, CX3CR1+ cells embark on a unidirectional migration and do not cross back into the intestinal tissue [29]. To prevent pathogens from traversing the epithelial barrier is a key goal and the rapid protective response mediated by CX3CR1+ macrophages is followed by an additional defensive strategy that restricts the replicative niche established by Salmonella that have managed to infect the intestinal mucosa. Pathogen-mediated CX3CR1 transmigration reached its peak after 5 h and
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declined at 12 h post-infection [29]. At a later time point, Sellin et al. [44] showed that, starting from 8 h post-infection, epithelial EpCAM+ cells harboring Salmonella could be observed in different stages of dislodging from the mucosa and are eventually expelled into the intestinal lumen. Mechanistically, the expulsion of infected IECs is driven by an epithelium-intrinsic defense axis dependent on the NAIP/NLRC4 inflammasome. Further quantitative and timecourse analyses clearly demonstrated the contribution of this mechanism to the reduction of intraepithelial Salmonella load. Also, although the expulsion mechanism closely mimics the physiological process of IEC shedding on cell death, a much larger percentage of expelled IECs contained intraluminal Salmonella, demonstrating that the expulsion preferentially targeted infected IECs. Like CX3CR1 transmigration, this inflammasome-triggered expulsion is dependent on flagellin, a NAIP ligand, and on Salmonella’s ability to invade.
Concluding Remarks Intestinal CX3CR1+ macrophages are a multitasking cell population that plays a central role in controlling intestinal immune homeostasis and barrier function and participates in mucosal defense and clearance. On infection with S. Typhimurium, these cells can discriminate between the different signals produced by the pathogens and, depending on the stage of infection and most likely on the geographical location in the gut, they can either extend TEDs to sample luminal antigens or migrate into the intestinal lumen to prevent Salmonella from traversing the epithelium. Dissecting in detail the underpinning regulatory signaling network will help us ascertain the respective roles of these two CX3CR1-mediated events in combating infections (see Outstanding Question). At this stage it is plausible to hypothesize that the transmigration of Salmonella-capturing CX3CR1+ macrophages is a rapid response that intervenes once the integrity of the epithelial barrier has been breached and that is part of a multilayered defensive strategy including preventive mechanical/biochemical barriers followed, if necessary, by additional measures (i.e., expulsion of infected IECs) to reduce the pathogen burden in intestinal tissue. Acknowledgments The authors thank P. Pople and U. Santosuosso for their help with computer artwork.
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Outstanding Questions What are the distinct microbial signals triggering antigen sampling or intraluminal migration by the same cell type? Also, given the different behaviors of CX3CR1+ macrophages in response to the same antigenic stimuli in geographically distinct areas of the gut, is there a local control that triggers these seemingly opposing functions? Finally, is the lack of Salmonellainduced transmigration in the colon due to an intrinsically different response of colonic IECs to Salmonella infection? Both indirect CX3CR1+ macrophagemediated sampling and migration have been studied so far in response to Salmonella. Are these Salmonella-specific events or do they represent a more general protective response to pathogens? During antigen sampling the body of CX3CR1+ macrophages remains in the LP and only the TED protrudes between IECs, whereas during migration the whole cell body migrates between IECs. Are different adhesion molecules/integrins involved in the dynamics of the two events? Is CX3CR1-mediated antigen sampling indispensable for mounting an antigen-specific response? Do intraluminal CX3CR1+ cells eliminate/neutralize pathogens by direct killing or by transporting them to areas of the gut characterized by a thicker mucus layer and more abundant microbiota that can have a deleterious effect on pathogen viability?
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